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MECHANISMS IN MOSQUITOES RESPONSIBLE FOR VARIATION IN 

SUSCEPTIBILITY TO INFECTION BY Dirofilaria immitis (LEIDY), 

ETIOLOGIC AGENT OF CANINE HEARTWOHM DISEASE 



> ; Y 
DONALD M. SAUEHMAN, JR. 



A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL 
OF THE UNIVERSITY OF FLORIDA IN 
PARTIAL FULFILLMENT 01 3MENTS 

FOR THE DEGREE OF DOCTOR OF 1'HILOSOPHY 



UNIVERSITY OF FLORIDA 
1980 



ACKNOWLEDGMENTS 

I would like to thank Dr. Jai K. Nayar, for the use of his 
laboratory and facilities and for his patience, guidance and support. 

I would also like to thank Dr. Richard Bradley, who was instru- 
mental in getting this investigation started, and Dr. Jerry Butler, 
who provided constant help in a variety of matters along the way. 

The support and patience of my wife and children were constant 
and boundless. 



ii 



TABLE OF CONTENTS 

Page 

ACKNOWLEDGEMENTS i 1 

LIST OF TABLES. . v 

LIST OF FIGURES . viii 

ABSTRACT ix 

INTRODUCTION 1 

General Rationale. , , 1 

The Pro M em.. 3 

SECTION I A SURVEY TO DETERMINE THE NATURAL POTENTIAL 
VECTORS OF CANINE HE ARTWORK DISEASE IN VERO 
BEACH, FLORIDA 5 

Introduction 5 

Materials and Methods 12 

Results and Discussion 20 

Conclusions 23 

SECTION II MECHANISMS IN_CX. NIGRIPALPUS AND AS. AEGYPTI 
RESPONSIBLE FOR VARIABLE PATTERNS OF SUSCEPTI- 
BILITY TO INFECTION BY D_. IMMITIS. ....30 

Literature Review 30 

Definitions and Usage .....43 

Materials and Methods 46 

Additional Procedures and Materials. 66 

Data Presentation and Statistical 

Analysis... 71 



iii 



Experimental Results and Discussion. ...74 

Culex Susceptibility Patterns - 

Background, 74 

The Effects of Hindrance of Migra- 
tion of Microfilariae to the Mal- 
pighian Tubules , .75 

The Relationship of Hemolysis and 

Crystal Formation to ousceptib.il- 

ity of Cx, nlgripalpus to Infection 

by D . immitia . 87 

Evaluation of Alterations in Sus- 
ceptibility Level of Cx. nigripal- 
pua to D. immitia infection induced 
by Additives in the Blood Meal.. 95 

The Determination of Sources of 
Microfilarial Vitality Loss........ 101 

Mode of Action of the Bucco- 
pharyngeal Armature, .....108 

Conclusions - Cx, nlgripalpus ....... 113 

Ae. aegypti Susceptibility Patterns - 
Background , , , . ............122 

The Effects of Hindrance Movement 

on Susceptibility of Ae. aegypti to 

D. immitis Infection 122 

The Effect of Atreptic (Nutritional) 
Factors , 1 24 

The Effect of Antiblastic Factors 124 

Hemolymph Factors. .....127 

The Chronology of Arrest of Development.. 128 

Conclusions - Ae. ae/?ypti . 130 

BIBLIOGRAPHY 1 57 

BIOGRAPHICAL SKETCH 168 



LIST OF TABLES 



Table Pag© 

1-1. Natural Potential Vectors of Canine Heartworra 
Disease in Vero Beach, Florida. Combined (35) 
Collections by Species, Trap Site and Trap Type. 132 

1-2. Natural Potential Vectors of Canine Heartworm 

Disease in Vero Beach, Florida. Species Collected 
and Species Found Positive for Dirofilaria immitis 
Infection 133 

1-3. Natural Potential Vectors of Canine Heartworm 

Disease in Vero Beach, Florida. Analysis of 16 

Positive Females From 3 Species 134 

II-'l.. Host Efficiency (Portion of Prelarvae in Tubules 
of Total Ingested Microfilariae) and Relationship 
Between Number Ingested and Prelarvae Developing 
at +24 Post Feed in Gulex ni.Tripalpus 135 

II-2. The Effects of Hindrance of Microfilarial Movement 
On Susceptibility of Cx. nigrlpalpus to D. immitis 
Infection Via "Normal" Bloodfeeding Mode, Measured 
by Mean (X) Prelarvae Per Female With Confidence 
Interval (CI) at PsO.05... ...... 1 3 6 

II-3. Comparison of Coagulation Patterns in Bloodmeals 
from Croups of Cx. nigripalpus Fed Directly On a 
Dog and Fed Via Membrane On Both Anticoagulant- 
Treated Dog Blood (AC) and Glass Bead-Defibrinated 
Dog Blood (GBDF), Tested by Coverslip Compression 
For Clot Presence * ^37 

II-4. Coagulation Times (Mean Minutes + Confidence Interval 
(Cl) at P=0.05) of 50 ul Samples of Untreated Dog 
Blood Added to 5 jil 0.596 NaCl Containing Dissolved 
Extract of Cx. nipyipalpus Salivary Glands 1 38 

II-5. The Effect of Midgut Extracts On Coagulation Time 

of 50 ul of Dog Blood 139 



Table Page 

II-6. In Vivo Hemolysis of Chicken Erythrocytes in 
Mosquitoes Fed Directly on the Host and Via 
Membrane on Anticoagulant-Treated or Glass Bead- 
Defibrinated (GBDP) Blood 141 

II-7. Hemolysis of Chicken Erythrocytes Following In 

Vivo, Non -Par pa Introduction of Cell Suspensions 

and In Vitro Teste on Extracts. 142 

II-8. Crop Contents Acidity: Distribution in Mosquitoes 
and Effects on D. immitis Microfilariae and Chick 
Red Cells 143 

II-9. The Effects of Alteration of the Blood Food Chemical 
Composition On Susceptibility_of Cx, ni^ripalpus to 
Q> immitis Infection. Mean (X) Prelarvae Per Mosquito 
and t 0.05 Confidence Interval (CI) and Susceptibility 
Prof il e 1 44 

11-10. [he Effects of Alteration of Maintenance Sugar and 

Blood Food Chemical Composition On Susceptibility, of 
Cx . nigripalpus to D. immitis Infection. Mean (X) 
Prelarvae Per Mosquito and t 0.05 Confidence Interval 
(CI) and Susceptibility Profile 146 

11-11. Microfilaria! Vitality; Tests for Antiblastic Factors 
in Cx, nigripalpus ; Mean (X) Microfilariae + Standard 
Error (SEj With Distribution According to Precent 
Moribund 3 47 

II-1 2. Microf ilarial Wound Assay. . , , 349 

II— 1 3- D. immitis Microfilariae Sucrose Tolerance Test .350 

II— 14. Hemolysis of Chicken Erythrocytes; Ingestion of 

Chicken Erythrocytes Via Sugar-Feeding Mode (BF-SF) 

Mean (X) With Standard Error (SS) of % Hemolysis 

Per Female and Hemolysis Profile. 351 

II— 15. Microfilaria! Vitality Patterns Microfilaria! 

Morbidity in Mosquitoes Infected Via Abnormal (Sugar 
Feeding) Mode. 352 



vl 



Table Pa * e 

11-16. Variation in Susceptibility Levels of Ae. aegypti 
to 2' i'T'^tis Infection. After Various Treatments 
tooupply Atreptic Factors or Delete Antiblastic 
Factors • 1 55 

11-17. Distribution of Mosquitoes According to otages of 
D. immitis . Harbored 120 h After Being Injected 
Into the Hemocoel, and Number and % Moribund of 
Larval Types and Number of Host Responses ...155 

11-18. Early Chronology of Arrest of Development of 

D. immitis Prelarvae in the Malpighian Tubules 

°f M* a e^yP t:i - ped Directly on the Infectious 

Dog, With An. quadrimaculatus Procedural Controls 

(PC), as Measured by Portion of Normal and Abnormal 

Prelarvae With the Passing of Time. Mean (X) + 

Standard Error (SE) With Percent {%) 156 



vii 



LIST OP FIGURES 

Figure 

1-1. Map of Vero Beach, Florida, shoving survey trap site 15 

1-2. Modified, high-survival, CDC-light trap receptacle 17 

II— 1 . Feeding methods: direct feeding of mosquitoes on legs 

of dog inserted through access sock 48 

II-2. Feeding methods: direct feeding of mosquitoes on shoulder 

of dog laid down on top of cage 48 

II-3. Feeding methods: membrane-feeder, schematic 50 

II-4. Feeding methpds: membrane feeder in position on top 
of cage, with water bath reservoir (left) containing 
pump and thermo regulator • 51 

H-5. Malpighian tubule squash for prelarval count 57 

II-6. Malpighian tubule squash for sausages 57 

II-7. Special apparatus for blood feeding mosquitoes in 

the sugar feeding mode-schematic ° 8 

II-8. Serial sections of a) anterior midgut of Cx. nigrip- 
alpus and b) posterior midgut of Cx. ni^ripalpus. 
showing free nuclei from hemolyzed cells, and c) 
posterior midgut of Ae. aegypti , showing aggulination 
but no hemocolysis • ' 



11-10. Distribution of wounds in microfilariae from Cx. nig- 

ripalpus 

H-11. Melanization host response of Ae, aegypti to D. immitis 
in the malpighian tubule • • 

11-12. Hemacyte host response of Ae. aegrvpti to D. immiti3 

in the hemocoel «.....« 



: ! i 



II-9. Sections trough buccal cavity showing dorsal pharyn- 
geal valve (bucco-pharyngeal armature) 115 



,106 
.126 
,129 



viii 



Abstract of Dissertation Presented to the 

Graduate Council of the University of Florida 

in Partial Fulfillment of the Requirements 

for the Degree of Doctor of Philosophy 



MECHANISMS IN MOSQUITOES RESPONSIBLE FOR VARIATION IN 

SUSCEPTIBILITY TO INFECTION BY Dirofilaria immitis (LEIDY), 

ETIOLOGIC AGENT OF CANINE HEARTWOHM DISEASE 



By 
Donald M. Sauerman Jr. 
December, 1980 



Chairman: Jerry Butler 

Co-chairman: R, E. Bradley 

Major department: Entomology and Hematology 



Based on the hypothesis that the susceptibility level of a potential 
mosquito vector to filariid nematode infection is determined by the presence 
or absence of physiological or morphological factors that manifest 
themselves as barriers at either the midgut or specific target tissue 
level, the patterns of susceptibility and mechanisms responsible for these 
patterns were investigated by employing an addition: deletion strategy 
for experimental delineation of mechanisms, using model mosquitoes to 
represent 3 frequently encountered susceptibility categories. The 
mosquitoes, Culex ni/yripalpus for partial susceptibility determined by 
a gut barrier, Aedes aegypti for partial susceptibility determined by a 
specific target tissue barrier, and Anop h eles quadrimaculatus for 
susceptible control, were chosen for their ability to sharply display 
the attributes of their particular pattern. 



ix 



Neither the variability of particular patterns nor the mechanisms 
that generate them have "been adequately explained or, with few exceptions, 
thoroughly studied. It was the aim of the research reported in this 
thesis to more clearly characterize these factors, since the variability 
of the patterns suggest that this might be an aspect of the vector-parasite 
relationship that is vulnerable to manipulation by man. A preliminary 
survey for natural potential vectors implicated 3 species, Aedes 
taeniorhynchus , Qui ex nigripalpus and Culex quinquefasciatus , as most 
likely transmitting the disease in the Vero Beach, Florida, area. 

Susceptibility level was determined in Cx. nigripalpus to be a 
function of 2 factors, hindrance of microfilaria! movement through the 
midgut by rapid clotting of the bloodmeal and wounding of microfilariae; 
the number of microfilariae ingested by Cx. nigripalpu3 fed on a highly 
microfilaremia (approximately 75 microfilariae/ul blood) dog was neither 
a factor in the determination of nor could be useful as an estimator of 
the susceptibility level. The addition of anticoagulants to blood fed 
to Cx. ni gripalpus via a membrane feeder failed to alter the susceptibility 
pattern, probably because they failed to prevent coagulation. However, 
the absence of salivary anticoagulant was not a factor, as extracts of 
salivary glands prevented clotting of venous blood. The presence of a 
powerful coagulant in midgut extracts was demonstrated, and, together 
with the production of shifts in the susceptibility pattern produced 
when Ox. nigripalpus were fed on glass bea.d-defibrinated blood, support 
the hypothesis that hindrance of movement of microfilariae is at least 
a partial determinant of susceptibility in the species. 



Hemolysis and crystal formation were associated with, but not 
responsible for, microfilaria! mortality in the Cx. nigripalpus midgut. 
Non-wounded but moribund microfilariae found in crops were probably the 
result of flora-produced low crop contents pH. Wounding, which occurred 
only in Cx. ni^ripalpus fed per os or sugar mode with plasma added, was 
demonstrated to be the other major determinant of susceptibility level, 
with the rapidly oscillating bucco-pharyngeal armature being the most 
likely source of the wounds. The involvement of the food-routing switch 
mechanism seemed likely, based on wound pattern variation associated 
with feeding mode, and explains shifts in susceptibility produced in 
mosquitoes fed on buffer-treated blood. 

Conventional addition: deletion strategies were non-productive in 
revealing the mechanism in Aedes ae,aypti that produces arrest of develop- 
ment of prelarvae in the tubules, and hindrance of movement was not a 
factor. While the melanization inhibitor, reduced glutathione, has 
some effect on arrest of development, only interval-feeding significantly 
altered the pattern. The phenomenon starts to occur within 6 hours of 
ingestion and has a chronological aspect. The intracellular-3tation 
hypothesis is not supported by the finding here of developing forms in 
the hemocoel after injection of microfilariae. 



xj 



INTRODUCTION 

General Rationale 
The fundamental rationale for the research in this dissertation 
is to support man's effort to control filariasis in his own kind and 
in his animals. Freedom from disease is requisite for the efficient 
use of our resources, which can translate into issues as basic as the 
success of birth control programs in populations that are breeding 
rapidly simply because the chance for any one child 1 s survival is so 
small. The breaking of the epidemiological triad can be attained by 
assaults on any of its links, and often involves a variety of approaches, 
as with filariasis, for example, which is attacked with assaults on the 
vector with insecticides and treatment of hosts with paraciticides. 
Treatment of the definitive host, either prcphylactically or thera- 
peutically, has certain advantages and limitations depending on the 
particular disease, but is definitely in ascention at the present time 
as one of man's major strategies against disease. In many diseases, 
the vector is placed under assault by man using the weapons of pest 
control, such as pesticides, insect growth regulators, genetic methods 
(sterile release; gene insertion), and integrated pest management. 
As with treatment of the definitive host, this strategy's weapons have 
their advantages and limitations. Our traditional weapons, the 
pesticides, have been shown by our own scientific community to be more 
accurately labelled "biocides," targetless weapons that have had a 

1 



way of coming back to man full-circle. However, judicious use of 
scientifically established levels of biocldes is still one of the 
components of modern pest management weaponry. Growth regulators and 
genetic methods are likewise limited to particular situations. Of 
oourse, the development of these techniques is on-going, with constant 
pressure for expansion of application for each system. Integrated 
pest management is likewise only just surfacing as a utilized concept, 
and awaits only the refinement of its ecological data acquisition 
strategies to expand its presently limited applicability. And, 
setbacks in systems, such as development of resistance or unacceptable 
contamination levels, add to the total cost of a system already 
expensive because of high development cost, itself the result of 
labelling procedures deemed necessary to prevent further contamination 
of the environment or the introduction of unseen health hazards. 

The satisfying of man's need as viewed from the standpoint of the 
public health researcher means solving epidemiological problems. Basic 
research serves as the breeding ground of applied technologies, a 
relationship that is fundamental in the generation of solutions since 
a thorough knowledge of a disease usually precedes its control. In 
basic epidemiological research, the concept of the triad is often 
utilized to conceptualize the interrelatedness of elements. The 
arrows connecting parasite-to-vector, vector-to-host and host-to- 
parasite imply a complexity of elements, since the arrows travel in 
both directions! for example, vector-parasite relationships involve 
both effects of the vector on the parasite and effects of parasite on 
vector, which is logical, since the vector is a host. 



One aspect of the epidemiological problem in the mosquito- 
onchocercid relationship that could prove to be of profound importance 
to the attainment of the thorough knowledge needed to conquer these 
diseases is the variation in susceptibility of potential vectors to 
the disease. The mechanisms that allow one mosquito to carry huge 
filariid larval loads while rendering others totally refractory have 
just begun to be investigated, with the first research specifically 
aimed at resolving this problem being less than 30 years old. It is 
the purpose of the research reported in this dissertation to Increase 
our knowledge of these mechanisms., 

T he Problem 
The susceptibility to filariid diseases in mosquitoes, while 
variable even at the individual level in some species, and from 
strain to strain in others, nevertheless falls into a limited number 
of generally valid categories, based on success in supporting larval 
development and transmitting the infectious stage. Most mosquitoes are 
either susceptible* partially susceptible or refractory as inter- 
mediate hosts, indicating that host specificity, perhaps the mo3t 
vulnerable link in the parasite's life-cycle for man to exploit, 
is involved in the production of these different susceptibility levels; 
characteristic patterns of susceptibility fall into as few as 6 or 7 
groups. 

The physiological mechanisms responsible for generation of these 
groups is poorly understood in most cases, although much has been 
learned. Two major barriers to successful development of parasites 
have been described in general terms for filariids, the gut barrier 



and specific host tissue barrier, although, in general, only one 
type of barrier exists in any one group. 

For this study, mosquitoes were chosen on the basis of their 
suitability as representatives of a particular type of susceptibility 
pattern, and this, in turn, usually resolves itself, physiologically, 
into a confrontation with one of the barrier- types mentioned above. 
The models possessed clear and sharp expressions of the patterns 
induoed by the mechanisms, increasing the chance of detecting experi- 
mentally-induced dislocations in the vector-parasite relationship. 
This, in turn, will lead to the characterization of the underlying 
mechanisms which are the genetically-determined transducers of suscep- 
tibility-level determinants. 

Prior to initiating laboratory experimentation, a survey for 
potential vectors of canine heartworm disease in the study area was 
conducted. The survey provided "hands-on" experience with the prob- 
lems inherent in such epidemiological studies, as well as exposure 
to the business-end of disease transmission. The result of the 
survey and its relevanoe to susceptibility studies is discussed in 
the first section, followed by the laboratory studies. 



SECTION I 

A SURVEY TO DETERMINE THE NATURAL POTENTIAL VECTORS 
OF CANINE HEARTWORM DISEASE IN VERO BEACH, FLORIDA 



Introduction 

Since 1900, when together Grassi and Noe first implicated mos- 
quitoes as intermediate hosts of Dlrofilaria immitis , the etiologic 
agent of dirofilariasis, or canine heartworm disease, a succession of 
researchers have sought to establish which species are actually in- 
volved in transmitting the disease. With few definite exceptions, 
canine heartworm disease is nearly as widely distributed throughout 
the world as its primary definitive host, while very few mosquito 
species are that cosmopolitan, exploiting, as they do, but also re- 
stricted to, a variety of habitats that may or may not bring them 
into the epidemiological web. Therefore, the determining of vector 
species when the disease occurs can be a local or geographic regional 
problem. The disease is characterized by its emanation from nidi of 
variable size, from neighborhoods to entire cities, and the intensity 
of the disease, measured by incidence in dogs or wild mosquitoes, is 
a function of the optimizing of the requirements of the epidemiological 
triad, which are rigorous. The infectious dog must occupy the same 
time and space as the bloodseeking and susceptible mosquito. If the 
mosquito is to vector the parasite, it must first survive the infection 
itself and then survive long enough (about 2 weeks) to allow parasite 
development, after which a second or third bloodmeal taken on a dog or 

5 



other suitable definitive host completes the cycle. That canine 
heartworm disease is endemic on the South Atlantic iind Gulf coasts 
of the United States attests to a confluence of satisfied epidemiological 
requirements, including host and vector density and the surmounting of 
array of further biological barriers. But nidi have been developing 
throughout the country (Harrison e t al. , 1965; Otto, 1972), probably as 
a result of human population mobility, with the result that formerly 
"clean" areas are now diseased; one often-cited example is California, 
where the first autochthonous cases were reported in 1970 (McGreevy 
et al. , 1970). 

One of the basic requirements for vectorship is susceptibility 
to the disease. That mosquitoes vary in susceptibility to D. immitis 
was first reported by Feng (1950) in Peiping, China, and this was 
quickly followed by the investigation of IIu (1930 in tne United 
States, who listed 12 species in J genera from 4 regions as known 
susceptible hosts for D. immitis , while determining the susceptibility 
of 8 species himself. Hu concluded that a good host species is one 
which shows a high group percentage of infection and high individual 
intensity of infection; he addressed susceptibility variation directly 
and was one of the first investigators to comment on individual variation 
within a species, an important milestone. In the same decade, Roubaud 
(1937) reported geographical strain variation in Aedes aegypti 's 
susceptibility to D. immitis infection. Further reports on potential 
vectors of CHD came from as far as the Phillipines (Rosario, 1936); 
and Yen's (1938) basic study was the first to establish clear-cut 
categories into which most mosquitoes could be placed based on their 



susceptibility. The physiological basis of susceptibility variation 
was finally addressed by Kartman (1953a), in a classic investigation 
attempting to link susceptibility with hindrance of migration and 
effects from digestive secretions. The determination of experimental 
vector potential has continued to be a basic method used to estimate 
the importance of a species in a particular area in transmission of 
CHD. These investigations include those conducted by Bradley (1953), 
Symes (i960), Beam (1965), Bemrick and Moorhouse (1968), Chellappah and 
Chellappah (1968), Weiner and Bradley (1970), Suenaga (1973, 1975, 1978), 
Intermill (1973), Weinmann and Garcia (1974), Seeley and Bickley (1974), 
Nayar and Sauerman (1975), Jankowski and Bickley (1976), Christensen 
(1977), and Rogers and Newson (1979). As early as 1970, over 60 
species, worldwide, had been proven to be "susceptible" to D. immitis 
infection (Ludlam et al. , 1970). The significant feature which emanates 
from these studies taken collectively is that to a greater or lesser 
degree, and with varying patterns, a large number of mosquito species 
are susceptible to D. immitis and have therefore satisfied a basic 
requirement for vectorship. 

In conferring potential veotor status, susceptibility determination 
can be definitive without further investigation if a particular species 
is solitary in the diseased area, and if increased rates due to immi- 
gration of previously infected dogs can be ruled out. However, there 
are more commonly several candidate species that qualify as far as 
susceptibility alone is concerned, and additional information must be 
acquired before ranking the species regarding relative vector potential. 
As Rosen (1954) points out, "In nature, the importance of a given species 
as a vector may be influenced by such factors as its abundance, the 



frequency with which it feed© on vertebrate hosts of the parasite, and 
its susceptibility to adverse effects of the developing filarial larvae. 
These factors may be of equal or greater importance than its relative 
susceptibility to infection" p. 525- Ecology and biology are important 
factors (Ludlam et al. t 1970), and these Include breeding sites, molts 
per year, longevity, flight range and, certainly, strength of host 
preference. There is fairly universal agreement that there is not 
enough information on natural infection and transmission (Ludlam et ai. , 
1970; Christensen and Andrews, 19?6). Ideally, such studies would 
require a high level of enzooticity, enabling the capture of enough 
infected mosquitoes to allow a "differential" count to be made. Further- 
more, experimentally determined susceptibility levels may differ 
radically from those measured in field populations, implying the 
presence of barriers in nature that do not manifest themselves in the 
laboratory. Thus, noting that only 1 of 466 field collected Anopheles 
punctipennis harbored developing forms while none had infective stages, 
both patterns radically different from experimentally-determined 
susceptibility levels for that species, Christensen and Andrews (1976) 
concluded, "It is possible that the parasite is unable to develop to 
the infective stage under natural conditions or may cause a high degree 
of mortality with this particular strain of Anopheles punotipennis" p.279« 
Furthermore, species are subject to elimination from consideration as 
vectors because of obvious mitigating faotors, such as absence of geo- 
graphic sympatricity, chronological confluence, rigid host preferences 
(rarely), and other often less obvious factors barring the mosquito 
from candidacy. Such factors, as host preference, are commonly used to 
explain absence of infection in a particular species (Christensen and 



Andrews (1976) with Culex tarsal is , for example), but host preference 
can he a matter of host availability or population density (Edman and Taylor 
1968), and eliminating from candidacy a species because it is rural 
is risky since dispersal or migration can suddenly bring it into 
contact with population centers and the epidemiology of the disease. 
Likewise, while number of molts can be a factor, it is probably less 
important than longevity and ecological focusing, important factors 
in establishing a restricted nidus with species 3uch as Ae. sierrensis 
(Weinmann and Garcia, 1974), while a short-lived mosquito, regardless 
of the extent of multivoltinism, is a poor candidate. Longevity is 
also altered by the effects of the parasite on the host, a phenomenon 
addressed by several investigators, and mortality in infected mosquito 
populations can soar above 90% long before parasite development is 
complete (Weiner and Bradley, 1970). 

The isolation of infected mosquitoes in nature obviates the 
acquisition of much circumstantial evidence. Confluence of host and 
vector has occurred without the artiface3 of the laboratory. If there 
are developing forms, she is susceptible without further ado, and if 
L3 infective stages are found in the head and proboscis, her candidacy 
as a potential vector, and thereby that of her species, is greatly 
strengthened. Host preference studies are made moot, and the ranking of 
the species in the population of all mosquitoes is possible. The 
only step untaken is proof of ability to actually experimentally 
transmit from dog-to-dog, which has been done, but only for a compara- 
tively few species, namely, Ae. to goi (Kume and Itagaki, 1955), An. 
quadrimaculatua (Newton, 1957), Ae» aegypti (Taylor, i960), Ae. vigilax 
(Bemrick and Moorhouse, 1968) and 4e t capariftnaia (Blcklev et al. t 1977). 



10 

However, transmission studies, as experimental susceptibility studies, 
do not demonstrate the natural act, only the potential for its occur- 
rence. 

The "paucity" of data on natural infection rates in mosquitoes, 
and the need to acquire it, has been addressed by Ludlam et al. . 
(1970), and they feel that it is attributable to the work involved in 
collecting, identifying and dissecting numerous specimens to obtain 
meaningful rates. With all other things being equal, increases in 
infection rates are attributable to an increase in number of infected 
dogs or a decrease in the number of diluting non-infected mosquitoes. 
Yields of infected mosquitoes from collections made with a variety of 
methods are often less than 2.1^? for example, Rosen (1954) collected 
and dissected over 4000 mosquitoes, representing 7 species, but only 
10 were positive in 2 species. Two species (Ae. aegypti and Cx. 
quinquefasciatus ) positive for susceptibility in experimental tests, 
were refractory. Similarly, a collection of 4746 mosquitoes representing 
19 species yielded a meager 3 mosquitoes with 1st stage larvae and 
prelarvae in 2 specimens, all from one species (Ae. vexans) (Bemrick 
and Sandholm, 1966) and of 2312 Cx. quinquefasciatus collected, only 
1.45% contained developing heartworm larvae (Villava30 and Steelman, 
1970). In field studies supplementing lab studies, engorged-only 
mosquitoes collected from a sentry dog complex where nearly one-half 
of the animals were suspected of being infected, infective L3 from 
individually dissected mosquitoes were found in 15.3% of one species, 
0.53% of another and none in 4 other species (Intermill and Frederick, 
1970). Suenaga (1973, 1975» 1978), in three separate studies conducted 
in different cities, determined natural infection rates ranging from 



11 



0.3% to 5.5% in 9 mosquito opecies trapped with human and dog bait 
traps or with light traps. Of 342 specimens representing 10 species 
collected at a kennel with a record high incidence of diseased dogs, 
only 4 infections were found in 3 species (Bickley et al. , 1976), 
and D. iiamltis was recovered from 4*96 of Ae» trivittatus , 0.42% of 
An . punctipennis (Christensen and Andrews, 1976), 0.03 - 0.82% in 4 
species of 2 genera in Malaysia (Easio and Cheong, 1977) • Eleven infected 
mosquitoes representing 3 species were found in a collection of 3445 
specimens (Arnott and Edman, 1978) and 1.4% of 3294 mosquitoes collected 
in Connecticut were positive for D. immitis (Magnarelli, 1978), while 
a rate of 0.21% was obtained from determinations of 25 pooled species, 
92 D. immitis being found in 3 species from a collection of 43*627 
individuals (Lewandowski et al. , 1980). Thus, even when conditions 
are optomized for finding infected mosquitoes, the rates are low and 
ranking by rate therefore difficult, and a major consideration in trap 
site or collection area selection must be yield if meaningful results 
are to be obtained (Ludlam e t ...al . , 1970). 

While it is certainly of concern that D. immitis is succeeding in 
establishing sylvan reservoirs in wild animals, with microfilariae 
having been discovered in the blood of wolverines (Williams and Dade, 
1976) and sea lions (Taylor e t al. , 1961), of greater immediate concern 
from the standpoint of human public health is the occurrence of heart- 
worm disease as a zoonosis. While the other dirofilariids associated 
with man's domestic animals ( Djrofilaria repens ) or closely impinging 
wild animals ( Dirofilaria tenuis in the racoon) are of little medical 
concern outside of dermatology since they are nearly always found in 
subcutaneous tissue, D. immitis is less well-adapted to man, and after 



12 

some development, probably in the right heart, it lodges in lung 
arterioles and dies; it is then thrombocized, producing pulmonary 
infarctions which, while largely symptomless, can cause a variety of 
symptoms from cough to chills to fever, and can, more importantly, 
lead to misdiagnosis as early bronchial carcinoma (Dissanaike, 1979) i 
which is undoubtedly stressing on the patient. Since the 68 cases 
reported during the last 2 decades have come from only 3 countries 
(USA, Japan, Australia), and 70% of the cases are symptomless, found 
by accident during radiography, the tip-of-the-iceberg phenomenon is 
probably occurring (Dissanaike, 1979; Weinmann and Garcia, 1974). 

Human pulmonary dirofilariasis has been described as a disease 
which "affects individuals living near coastal breeding areas" p. 407 
(Robinson et al. , 1977), and, indeed, most of the cases from the 
United States have been reported from Florida. With the immense 
increase in canine heartworm disease in Florida, with 493 reported 
cases in 1966, 1,250 in 1970, and 5»558 cases in 1976 (Florida 
Morbidity Statistics, 1966, 1970, 1976), the determination of natural 
potential vectors and their rate of infection constitutes an important 
prerequisite to a full understanding of the disease. The purpose of 
the present survey is to determine the natural potential vectors of 
heartworm in a region of Florida highly endemic for heartworm and 
therefore where introduction into humans is undoubtedly commonplace. 

Materials and Methods 

The basic strategy for the acquisition of data needed to determine 
the natural potential vectors of D. immitis is to collect wild mosquitoes 
from suspect areas. Conducting studies where there is an "adequate" 



13 

source of infectious dogs is requisite (Ludlam et al. , 1970). Field 
researchers have made use of a variety of devices and procedures to 
more or less maximize the yield of infected wild mosquitoes; however, 
the aim of the investigation is what in the final analysis dictates 
choice of collecting devices and the sites where they will be put to 
use. One such survey restricted collections to within 40 yds. of 
human habitation (Rosen, 1954) » while others have U3ed established 
dog yards (Villavaso and Steelman, 1970), kennel walls in kennels 
harboring heartworm infected dogs (Intermill and Frederick, 1970), 
hunt clubs with infected dogs (Bickley et al. , 1976), areas with 
reported heartworm disease with breeding areas nearby (Arnott and 
Bdraan, 1978) and sites of recent infection (Lewandowskl et al.. 1980). 
While most collection sites are intentionally established in residential, 
suburban areas, to capitalize on confluent dog and mosquito populations, 

2 investigations (Villavaso and Steelman, 1970$ Suenaga, 1973) utilized 

3 distinct human (and, therefore, dog) habitats, and the results in- 
dicated that while residential areas are in general predictably higher 
in the number of infective mosquitoes, other factors than dog density, 
such as proximity of breeding sites, especially for domestic mosquitoes, 
and owner habits regarding leaving of pets outdoors and unprotected, 
influenced the yield of infected mosquitoes. 

As there is variation in collection site depending on the aims of 
the investigator, there is similar variation in the device selected to 
sample the suspect population. Biting collections have been used by 
some (Rosen, 1954; Arnott and Edman, 1978), aspiration of engorged 
females by others (intermill and Frederick, 1970), while more commonly 
used are light traps (Suenaga, 1973, 1975 » 1978; Bickley et al. , 1976; 



14 

Christensan and Andrews, 1976; Arnott and iidman, 1978; Lewandowski 

et al. , 1980) and bait traps, usually dog-baited (Benirick and Sandholm, 

1966} Villavaso and Steelman, 1970; Suenaga, 1973; Lewandowski et al. , 1980). 

but sometimes human-baited (Suenaga, 1973). Rosen (1954) collected 

from pigs, humans and dogs, while power aspirators were used to collect 

resting mosquitoes from the leaf litter (Arnott and Edman, 1978). 

Thus, there are no hard-and-fast rules for making epidemiological 
surveys, except that the goal is to find the mosquitoes that are in- 
fected, and to find them in sufficient numbers to allow their being 
ranked by infection rate. In this study, which was conducted from 
April to August, 1977 » 3 sites (Figure l) and 2 trap types were 
employed. Site 1, in northeast Vero Beach, was in a residential area 
closest (1.5 km) of the 3 sites to the Indian River salt marshes. Site 
2 was in the center of the city's residential area, about 2 km further 
west than site 1 from the river. Site 3, in southwest Vero Beach, was 
about 4 km from the Indian River, but considerably closer to rural, 
agricultural and ranch areas, without the insulating barrier of houses 
extant at site 2. Thus, while all 3 sites had substantial dog popula- 
tions (personal canvass), the proximity to breeding areas, other than 
those normally found near homes, such as containers and bird baths, was 
considerably lower for site 2, while sites 1 and 2 were considerably 
further from salt marsh. Owner habits towards pets were not determined, 
but there was no subjectively distinct difference between the areas in 
types of housing or lifestyle. 

Mosquito populations at each site were sampled using 1 light 
trap (CDC-type) and 1 bait-trap (inaccessible chick-baited, lard can 



15 




Figure 1-1. Map of Vero Beach, Florida, showing survey trap sites. 
Scale: 1cm = 0.51km 



16 

trap as used by Nayar (1980), both with carbon dioxide adjuvant. Sawn 
blooks of dry ice, 5-6 cm on a side, were placed in 2.54 cm thick 
styrofoam boxes, made specifically to hold the blocks, tied shut with 
string and suspended a few inches above each trap. Traps were suspended 

from available vegetation, not more than 1 m from the ground. They 

h h 

were set out approximately 1 before dusk and collected at 0800 the 

following morning. In both traps, captured mosquitoes had access to 
10% sucrose solution, and the collection container for the light traps 
was modified to enhance survival (Figure 1-2). After returning to the 
laboratory, each collection was lightly chloroformed and placed in 
high humidity chambers with access to I0,i sucrose solution until 
dissected. Dissection was initiated as soon as all collections had 
been removed from the traps, with each sample anesthetized with chloro- 
form and separated according to species. Then, one 3pecies at a time, 
heads were removed to small saline droplets on siliconized slides; as 
many as 30 heads/proboscides could be processed individually per slide 
by placing a 24 x 50 mm coverslip over the droplets and evenly compres- 
sing the heads. The alimentary tract, including Malpighian tubules, 
was drawn from the body of the mosquito into a droplet of saline by 
grasping subterminal segments with insect forceps and slowly pulling 
backwards while holding the rest of the body. The midgut was discarded 
if no bloodmeal remnants were observable under a dissecting scope; all 
normal-appearing tubules were subjected to a mild coverslip compression 
as with heads, allowing individual inspection, but with less pressure, 
unless there were larvae visible or there was obvious pathology (swollen 
areas, clear areas) in the tubules, in which case more delicate compres- 
sion revealed the stages within the tubule without disrupting them. 



17 



to light trap 



sugar vial hole 




cloth holding ring 



'•*— 86mm -* 1 




jitmm 



sugar vial 



\j&&\rtflom cloth 

r, U v 

£1 * t 32mm 



Figure 1-2. Modified, high-survival, CDC-light trap receptacle. 



18 

Generally, prelarvae and early sausages are not easily detectable 
without coverslip compression, as they are intracellular, and the 
tubules must be considerably compressed; such compression easily 
destroys beyond recognition the later stages, especially stage II. 
Midguts with bloodmeals were ruptured in saline and examined for presence 
of microfilariae and type of red cell (nucleated vs. non-nucleated) 
if possible. 

Traps were set out and collections recovered twice weekly for 
nearly 4 months, yielding a total of 35 collections. Since each 
day's collection was composed of 6 traps, each individually processed 
by species, the time factor was considerable. Collections were dis- 
continued when breeding conditions improved, resulting in samples too 
large to process individually. 

In addition to processing-time problems, another source of concern 
regarding the results of such surveys is the identification of nematode 
larvae or their source. Thus, there are not only other dirofilariids 
in nature, but there are other animals than the dog with D. immitis 
infections (Levine, 1968). Since there is almost always possible 
contamination from other fiiariids, even when collecting engorged 
mosquitoes from kennel walls (Intermill and Frederick, 1970), that is, 
they could easily be seeking their 2nd or 3rd bloodmeal after having 
been infected with a different filariid on their 1st or 2nd, a con- 
siderable amount of judgment is needed to make a "presumed" identifica- 
tion, and this judgment must take into account the role that wild fauna 
might be playing. The rationale employed here are not "fool-proof" and 
offer a "presumed" identification in the fashion of other workers in the 



19 

field. The strength of the rationale varies, depending on the stage 
found and part of the mosquito from which it was recovered. Hach 
stage will be treated separately aa it relates to 4 criteria: 
l) measurements, 2) cuticular wrinkles, 3) caudal (not anal) papillae 
and 4) location in the mosquito: 

Microfilariae : The microfilaria is herein defined a3 that form still 
in association with the vertebrate blood, be it in the vertebrate 
bloodstream or invertebrate midgut bloodmeal; those have no potential 
for further development in thi3 association but must attain lodging in 
the target tissue, the Malpighian tubules in this case, prior to 
initiation of growth and development. The microfilaria is rejected as 
D. immitis if it is l) less than 4.8 p. in breadth or 283 u in length, 
which are characteristic of Dipetalonema reconditum (Newton and Wright, 
1956); or 2) greater than 327 u, as with Mrofilaria striata (Orihel 
and Ash, 1964), or greater than 370 ya, as with D. tenuis (Pistey, 1958)? 
3) or if it has cuticular wrinkles or striations, as in D. tenuis 
(Pistey, 1958). 

Prelarvae : The prelarvae is essentially identical to the microfilariae 
except that it is now in the specific target tissue, the Malpighian 
tubules. Only dirofilariids use this site, and thus prelarvae of 
Dipetalonema are automatically eliminated from consideration while 
D. tenuis and D. s triata (highly improbable anyway, if the survey by 
Orihel and Ash, 1964, is taken into consideration) are delineated as 
for MP. 

Developing Forms : Absence of information that would allow precise 
identification of these forms again precludes precise identification, 
but the only probable contaminant is D. tenuis , which has characteristic 



20 

cutlcular wrinkles, especially prominent in these stages, that allow 
it to be differentiated from D. immitis (Pistey, 1958). 
L3 : when found in the tubules, only 1). tenuis is a probable candidate 
other than D. immitis , which is rejected if cuticular wrinkles are 
present. When found in the head: proboscis, D. immitis is rejected if 
the length is greater than 1250 u, most likely Setaria (Becklund and 
Walker, 1969), or width less than 17;*, (possibly Foleyella spp.) 
(Benach and Grans, 1975; Kotcher, 1941), or greater than 28 ;i, possibly 
Mpetalonema . Absence of caudal papillae eliminates all Foleyella and 
possibly D. tenuis , while Mpetalonema reconditum has a long tail with 
2 subtenainal, ventrolateral ears, much more prominent than the nearly 
invisible nubs on D. immitis . It should be noted that Orihel (195 9) 
was unable to delineate D. tenuis from D. immitis ; therefore, the criteria 
used here in that regard, especially for L3, are not outside of debate. 

Furthermore, rejection of D. striata from the bobcat, based on a 
study by Orihel and Ash (1964) who found no D. striata in any of 15 
cats dissected, is not without risk, although it should be pointed out 
that the cats examined were from central and south Florida. Dipetalonema 
arbuta from rodents is less than 18 u in breadth and has 3 caudal papillae. 

Results and Discussion 
Thirty-five collections of mosquitoes, each comprised of 3 light 
traps and 3 bait traps, a total of 210 trap samples, yielded 1030 
specimens representing 13 species in 5 genera (Table 1-1 ). Three 
species, Cx. nigripalpus , Ae. taenlorhynchus , and Cx. qulnquefasciatus , 
accounted for 7#o of the total collected, with no other single species 
constituting more than 5%. While comparable numbers of Cx. nigri palpus 



21 

and Ae. taeniorhynchus were collected by both types of traps, bait 
traps accounted for a vast majority of Cx. quinquef asciatus . However, 
possibly due to the use of CO adjuvant with both systems, the contri- 
butions made to the total collection by both types of traps were 
comparable (46% for light traps and 54% for bait traps). As might 
be expected, based on its proximity to the Indian River salt marsh 
area, site 1 collections accounted for nearly twice as much (58%) of 
the Ae. taeniorhynchus collected than the next largest site, site 
3 {30/o), Similarly, site 3, closest to the rural ranch-grove areas, 
accounted for 60% of all Ox. nigripalpus collected. However, the sizable 
collections of Ae. taeniorhynchus at site 3 (79/264=30%) and of Cx. 
nigripalpus at site 1 (87/403=226) attest to the dispersal or migratory 
powers of these species. The large portion (122/200=60%) of Cx. 
quinquef asciatus at site 3 is likely a reflection of its proximity to 
residential sewer treatment systems, not found as a rule at the other 
2 sites, which are serviced by a central plant in the city system. The 
efficiency for the capture of each species at each site was, with some 
exceptions, similar to the rankings between sites, but slightly more than 
half of all mosquitoes collected were contributed by site 3 (526/IO3O), 
and only 16% (165/1030) by site 2, which contributed the fewest total 
mosquitoes of the 3 major species collected. 

The same 3 species that accounted for 76% of the total collected 
accounted for 100% of 16 nematode-parasitized mosquitoes (Table 1-2 ), 
2 of which (Cx. quinquef asciatus - 2 and Cx. nigripalp us - 3) were not 
infected but had ingested microfilariae in their bloodmeals, and one 
infected mosquito (Ae. taeniorhynchus ) with a non-identified L3 (Table 
I- 3 ). Thirteen presumed-J). immitis infected mosquitoes, or 1.3% of 



all collected (1030), bore a total of 101 larvae of all 4 stages, 
one mosquito (Ae. taeniorhynchus - 2) harboring both LI and L2. Only 
one mosquito (Cx. quinquefasciatus - 3) was found with prelarvae, 
indicating since they were active, that it had fed during the previous 
24-56 hours. The portions of other developing forms (LI and L2) and 
L3 are similar in pattern to groups fed by Kartman ( 1953a) f but this 
could easily have been coincidence, and the prelarvae rank is exactly 
the opposite of where it should be based on such a supposition. The 
percentages positive for the 3 species (Table I- 2 ) are not significantly 
higher or lower than those reported in the literature. Thus, 0.1% of 
Cx. quinquefasciatus were found infected by Rosen (1954), although he 
found none harboring L3; however, with Cx. annulirostris, he found that 
2.5% harbored developing fcrrci3. Basio and Gheong (1977) reported rates 
of infection varying from 0.03 to 0.82/j for 4 different mosquitoes, 
while an unusually high rate of 15.3% was reported for Mansonia 
uniformes (intermill and Frederick, 1970), although collections of 
engorged mosquitoes from sentry dog compounds undoubtedly facilitated 
improved rates. For Cx. quinquefasciatus , Villavaso and Steelman (1970) 
reported a range of 0.0% (rural yard) to 2.3/6 (suburban), and there was 
considerable variation within site types, depending on proximity of 
breeding places and habits of dog owners regarding leaving animals 
outdoors. 

It is provocative that 77/6 (IO/13) of all D. immitis infected 
mosquitoes were found at site 1 (Table 1-3 ), which accounted for only 
53% of all mosquitoes collected, compared with 51% for site 3, which 
contributed only 15% (2/13) of the infected mosquitoes. While site 1 
might be expected to have more infected Ae. t aeniorhynchus because 58% 



23 



of this species was found there, and the rate at site 1 for Ae . 
taeniorhynchus was 7/155 (l/22) or A*&A, the total Ae. taeniorhynchu3 
at site 3 was 79t making the expected number of infected Ae . 
taeniorhynchus at a rate of 4.6,'u at least 3 mosquitoes. However, 
the only other site with any infected Ae. taeniorhynchus was site 2. 
In a similar vein, site 3 accounted for 122/200 {61%) of all Cx. 
quinquef asciatus ; however, only one Cx. quinquefa3ciatus (3) was 
found there, infected with prelarvae, while 2 infected Cx. quinqu ef asc iatus 
were found at site 1 with only 49/200 (24.5%) of all Cx. qulnquefasciatus 
collected. Thus, the abundance of the species was not related to its 
infection rate, but seems, rather, to be site-dependent; site 1 is 
clearly contributing a disproportionate amount of infected mosquitoes, 
considering the numbers collected, or, conversely, site 3 is contributing 
far fewer than would be expected if rate was a function of collection 
size. To speculate for a moment, while site 1 was characterized in the 
Materials and Methods in terms of its proximity to the Indian River 
salt marsh, it also differs from the other 2 sites with respect to the 
East Florida Ridge, a limestone spine running down the east coast of 
Florida at varying distances from the Indian River or Atlantic Ocean. 
It is also within 0.5 km of a golf course with pond water hazards and 
much luxuriant, well-watered vegetation, thus providing both breeding 
and resting areas for Cx. quinquef asciatus and dispersing Ae. taeniorhynchus . 
Both other sites are west of that ridge, where the character of the flora 
differs from that east of the ridge, possibly as a function of humidity. 
Basio and Cheong (1977) saw that shady biotypes were more infectious. 
While no data were collected in this regard, it offers habitat-oriented 
alternative to the other salient possibility, mainly that there were more 



24 

infected doge at site 1. Remembering that the weather conditions were 
arid during most of the collection period, with rains coming toward 
the end, the survival value of the more moist habitat at site 1 could 
be extremely important, and offers an explanation for the observation 
that 80% (4 out of 5) of mosquitoes with L3, likely, but not prima 
facie , the oldest of the group, since it takes a minimum of 10 day3 to 
develop L}, were collected at site 1. (Of course, a very old mosquito 
can take its 1st bloodmeal and harbor only prelarvae on inspection). 
That the dry spell produced an older population from which the samples 
were drawn is fairly obvious. Not only was the population low, but, 
without the influx of young mosquitoes, especially Cx. quinqu ef asc iatus 
and Ae. taeniorhynchus , which would not benefit from grove irrigation 
practices as Cx. nigripalpus , the population would contain more and more 
mosquitoes of older and older age and thereby increase the chances, 
all other things being equal, of sampling an L5 infected mosquito, while 
the portion of such mosquitoes would be diluted by large numbers of new 
arrivals diluting lowering the average age. Survival rates in mosquito 
populations are very low under good conditions (Nayar, 1980), and under 
arid conditions, must be quite a lot lower; if the number of micro- 
habitats conducive to survival were greater in one area than another, 
it could be a major factor in disease transmission. 

Assessing the vector potential of mosquito species naturally 
collected but non-parasitized (equals negative results) is a common, if 
risky, practice, based a3 it is on circumstantial inference of widely 
varying strength, and no such assessment or .judgment will be made on 
this group here. However, since these inferential conclusions are 
often cited in the literature, a discussion of their merit and application 



25 

to the present work i3 in order. The following parameters have been 
used to support negative data (absence of infected mosquitoes from 
wild collections) or to rank those mosquitoes found naturally infected; 1. 
distribution (Rosen, 1954; Arnott and adman, 1978); 2. abundance 
(Rosen, 1954; Berarick and Sandholm, 1966; Ghristensen and Andrews, 
1976; Arnott and adman, 1978; Lewandow3ki et al. , 1980); 3. survival/ 
longevity (Rosen, 1954; Ghri3tensen and Andrews, 1976; Arnott and 
Edraan, 1978 — mentioned in text but not included in table; Lewandowski 
et al, , 1980); 4. vertebrate host preference (Rosen, 1954; Bemrick and 
Sandholm, 1966; Christensen and Andrews, 1976; Arnott and adman, 1978; 
Lewandowski et al., 1980); 5. survival with heavy parasite infection 
(Rosen, 1954; Bemrick and Sandholm, I966; Christensen and Andrews, 
1976); 6. multiple generations (Arnott and adman, 1978); 7« too small 
a sample (Christensen and Andrews, 1976; Bemrick and Sandholm, 1966); 
8. seasonal abundance (Bemrick and Sandholm, 1966; Arnott and adman, 
1978; Lewandowski e±_aL»» 1980); $. experimental vector potential 
(Rosen, 1954; Bemrick and Sandholm, 1966; Arnott and adman, 1978); 
10. natural vector potential and infection rate (Rosen, 1954; Arnott 
and Edman, 1978). In addition, mosquitoes have had their vector 
potential reduced or eliminated for l) insufficient numbers to be 
reliable vectors, erratic populations, distribution is erratio, 
not common, occurring sporadically later in the season, distribu- 
tion is pocketed, and late seasonal build-up (Arnott and Edman, 
1978). The strength of the judgment could be enhanced when different 
circumstantial lines of evidence are used in concert, as has often been 
done, rather than eliminating a species because of the presence or 
absence of one factor, unless that factor is ironclad. The problem 



26 



with this kind of assessment is that not all the parameters are 
equally valid, and yet it would score the same on a yes-no type 
ranking. For example, host preference is often cited, but this is 
parameter of varying value, from weak to strong, depending on the 
host and mosquito. The preference for bird blood shown by Culex spp 
is often only just that, a preference, and many Culex readily feed on 
mammals when mammals are abundant (Edman and Taylor,1968 ); thus, host 
preference can be related to host abundance. On the other hand, 
some mosquito host-preferences are more fastidious, such as with Gx . 
territans, which will feed on frogs and other amphibia (Edman, 1974). 
Most common mosquitoes, though, are blatant opportunists, including 
Cx. nigripalpus (Edman, 1974), and the literature is replete with 
reports of Culex feeding on dogs (see Villavaso and Steelman, 1970), 
so the "weight" given to a parameter such as host preference i3 variable 
and requires both knowledge and sense to insure its value to evalua- 
tions of vector potential. 

Likewise, it is not easy to determine what weight should be 
assigned voltinism, or number of generations. A relatively large 
number of generations is only important if it leads to an increase in 
population, and this iroreaae in population is related to vector 
potential. Since populations can go up or down with each succeeding 
generation, depending on the survival and number of eggs laid by the 
previous generation, the survival of which depended on environmental 
conditions extant during their lifetime (Nayar et al. . 1980), it is a 
difficult parameter to correlate with vector potential. Abundance 
itself may not be important if the vector is long-lived and is "ecological- 
ly focused " (Weinmann and Garcia, 1974) » while, for normally rural 



27 

species, such as Cx. nigripalpu3 , abundance might be requisite for 
vector potential, huge populations having to build up before sub- 
stantial, epidemiologically-important penetration of residential 
areas occurs. 

Seasonal fluctuations in susceptible populations can be of 
extreme importance in determining vector status. For example, in 
the present investigation, only 27 Ae* vexans were captured, and 
the very low rates of infection found in natural populations by 
Bemrick and Sandholm (1966), 1 infected Ae. vexans per 859 collected, 
mean that only extremely good fortune would have yielded an infective 
mosquito from the sample if the same rate operates here. However, since 
large Ae. vexans populations can rapidly be produced in groves during 
periods of irrigation, if the survey had been carried on, it might 
have included such a population and yielded infected individuals, if 
incidence in this mosquito is related to abundance. Essentially, what 
this implies is that sample-size can be very important, as noted by 
Christensen and Andrews (1976), and, unless rates of infection are 
extremely high due to focusing (Weinmann and Garcia, 1974), many 
mosquitoes will have to be collected before a reliable and reasoned 
judgment can be made. Thus, there is much more ground for Arnott and 
Edman's (1978) claim regarding the low potential of Ae. vexans than if 
the same claim were made for Ae . vexans here, because their sample size 
(410) was considerably larger, although their "Ho" ranking of Ae . 
vexans regarding field isolations ignores the work of Bemrick and 
Sandholm (1966), and no "longevity" or "survival after infected" column 
appears in their table of Biological Characteristics, although they 
themselves acknowledge its importance in the text. 



28 

During the interval during which this survey was conducted (April 
to August, 1977)* it can be concluded that Ae. taeniorhynchus was the 
major veotor of canine heartworm disease in the Vero Beach, Florida, 
area. Cx. quinquefasciatus was also an important vector and Qx » 
nigripalpus, playing much the same role as in St. Louis Encephalitis 
transmission, became entangled in the heartworm epidemiological web 
by invading residential areas. Ae. taeniorhynchus has many of the 
characteristics of Ae. trivittatus , which "carries very large numbers 
of parasites, yet is able to retain the necessary mobility to fly". P.. 278 
(Christensen and Andrews, 1976). Furthermore, the large developing- 
form loads in the tubules "suggests considerable tolerance by this 
mosquito to the damage done by the developing juveniles." If "tolerance" 
means survival, then the results are similar to those reported by Nayar 
and Sauerman (1975) for Ae. taeniorhynchus , where survival of infected 
mosquitoes for 15 days was 54%. The distribution patterns of CHD in 
the eastern United States (Otto and Bauman, 1959) strongly suggests 
salt marsh mosquito involvement. Ae. soil ici tans , the predominant salt 
marsh mosquito of the New Jersey and much of the northeastern coast, has 
already been shown to be susceptible to heartworm disease (Beam, 1965), 
although other studies (Nayar and Sauerman, 1975) indicate that its 
vector potential is less than that of Ae. taeniorhynchus because of 
poor survival (4%) of the infected mosquito. Survival after infection 
is of overriding importance to vector potential, and is one reason given 
for the low vector potential of Ae. aegypti (Rosen, 1954). As to whether 
Ae. taeniorhynchus would have been the dominant potential vector in a 
succeeding collection period, or at any other point in time, is not 
answerable by this study. Certainly, the seasonal fluctuations of mosquito 



populations generates an array of relative incidences of infected 
individuals of each species and possibly their rank in the order of 
potential vectors. 



SECTION II 

MECHANISMS IN CX. NIGRIPALPUS AND AE. ASGYPTI 
RESPONSIBLE FOR VARIABLE PATTERNS OF SUSCEPTIBILITY 
TO INFECTION BY D. IMMITIS 

Lite rat ure Revi ew 

That events occurring in the midgut of the arthropod host might 
determine the portion of onchocercid microfilariae that attain lodging 
in the target tissue and ultimately become infective was first suggested 
in experiments by Noe (1908) f who observed the death of microfilaria® 
of Dlpetalonema grassi in the gut of Rhipicephalus sanguineus , the 
brown dog tick, during the final phases of feeding. Given the length 
of time transpired since the initiation of feeding, it is not incon- 
ceivable that digestive processes were involved. The digestion of 
blood food by hematophagous insects is a complex subject, well-reviewed 
by Gooding (1972), who states as a rationale for comparative studies, 
"A second and more practical reason is that many blood-sucking arthropods 
are vectors of pathogenic organisms. Since most of these pathogens 
spend some time in the gut of the vector, digestive processes of the 
insect could influence vectoring ability" p. 5. Evidence in support of the 
role of the midgut in mediating the intensity of filarial infections 
in mosquitoes is suggested in the results of experiments by O'Connor 
and Beatty (1938), who found only dead microfilariae of Wuchereria 
bancroftl in a substantial number of Qui ex quinquefasciatus midgut 
bloodmeals. Some mosquitoes harbored substantial infections but many 

30 



31 

had "feeble" microfilariae. Similar results were described by Kotcher 
(1941), who observed that a majority of Foleyella microfilariae were 
killed in the gut of Cx. plplens and Cx. quinquef asciatus . This work 
was supported and expanded by Benach and Crans (1975)» who reported 
that only a small percentage of F. flexicauda penetrated the midgut 
of Cx. territans , with most microfilariae dead in the midgut shortly 
after feeding; the authors conclude, "Kventa occurring in the alimentary 
canals . . . appeared to be the major cause for differences in vector 
susceptibility" p. 209; further, that "there are strong indications 
that a gut barrier exists in Cx. territans which limits the number 
of microfilariae entering the hemocoel. The lysis of the frog 
erythrocytes and the death of the bulk of microfilariae, within a 
short time after ingestion, suggest that this barrier may be related 
to either the digestive secretion of the salivary glands and midgut, 
or to mechanical action of the bucco-pharyngeal apparatus, or both"p.211. 
Although reports of microfilaria! morbidity in the midgut are most 
consistent in Culex . essentially the same pattern of events apparently 
unfolds in the Anopheles gambla e:B rugia patai vector-parasite relation- 
ship, very few microfilariae reaching the thoracic musculature, with 
normal development of those that do (Laurence and Pester, 196l); 
Crans (1973)» comparing the susceptibility of An. gambiae and Cx . 
quinquef asciatus , reported similar results, with far greater suscepti- 
bility in the latter; differences in numbers ingested were not considered 
a mediating factor in susceptibility as measured by intensity of in- 
fection. In groups of Cx. q uinquef asciatus fed on heparinized blood 
infectious for B. pahangi , as much as 99»'7% of the microfilariae are 
still in the midgut at +1 , and, more importantly, this portion remains 



32 

essentially unchanged when measured at +24 (Ewext, I965); moreover, 
in terms of vitality, only one-fourth of the ingested microfilariae 
showed any activity at all, and thi3 was always feeble. Complete 
refractoriness to infection by B. pahan^i with dead microfilariae 
in the gut has been reported in the same mosquito (Ogunda, 1969); 
and, in the same system, using Cx. pipiens , Obiamiwe (1977) observed 
that 99.1% of the filariae failed to escape the midgut; 2 barriers, 
one relating to clotting time and the other to unknown factors, were 
mentioned as sources of the observations. Working with the unsheathed 
onchocercld, Dirofilaria repen3 , which does not penetrate the wall of 
the midgut, but rather migrates directly to the Malpighian tubules, 
Coluzzi and Trabucchi (1968) associated the death and injury of filariae 
to the mechanical action of the bucco-pharyngeal armature, anatomically 
known as the dorsal pharyngeal valve (Uchida, 1972). Dead larvae were 
associated with mosquitoes possessing this structure, which includes 
members of the subgenera Cellia and Kyssorhynchus of Anopheles and 
Culex of genus Qui ex (only 1 Culex was examined, Cx. pipiens ). These 
experiments were expanded by Bryan et al. (1974) and McGreevy et al. 
(1978), using B. pahan/?i and Wuchereria bancrofti with Cx. 
quinquefasciatus , An. ^ambiae and An. farauti , and Ae. togoi and 
Ae. aegypti . 

Similar patterns of susceptibility indicating the presence of a 
gut barrier have been detected in the U. immitia i Culex vector-parasite 
relationships, indicating again that such barriers are not parasite- 
specific. Evidence includes the observation by Peng (1930), that none 
of 31 Cx. pipiens examined was susceptible to 0. irnmitis , and the 



33 

comparative experiments of Hu (193*)» the firat researcher to discuss 
mechanisms that might explain susceptibility variation among3t species; 
he found "degenerate" microfilariae in Cx. pipiens one day after 
feeding, and concluded "Although it is a question what role physio- 
logical and hereditary factors may play in determining the variation 
in degree of resistence to infection with this parasite displayed by 
the mosquito hosts, they must be of some significance" p. 626. O'Connor and 
Beatty (1938) found large numbers of dead D. immitis microfilariae in 
the midgut-bloodmeals of Ox. quinquefasciatus bloodfed directly on the 
infectious dog, while Cx. tarsalis , Cx . t err i tans and Cx. pipiens 
vector-parasite relationships with D. immitis were all placed in the 
category of low intensity but supportive of normal development, in a 
scheme of susceptibility levels devised by Yen (1933), including J 
classes and 7 groups, based on intensity and percentage of infection. 
Yen concluded the question of why some species of mosquitoes are more 
suitable as the intermediate hosts than others appears to be unexplain- 
able by the morphological and taxonomical differences of mosquitoes, 
for even members of the same species vary to a considerable degree in 
their susceptibility to infection with the parasite, '//hat mechanism 
is involved in bringing about these variations in host susceptibility 
of the different species of culicid hosts is difficult to determine 
until we have a better understanding of the physiology and pathology 
of the insect. Travi3 (1947) found dead D. immitis microfilariae in 
Cx. quinquefasciatus . as Seeley and Bickley (1974) did in Cx. sallnarius . 

The earliest work in which the main focus of the study was to 
uncover mechanisms to explain the variation in susceptibility level 



34 

amongst species was Kartman 'a (1953a) investigation of the vector- 
parasite relation between JD. immltis and 3 mosquitoes, Ox. quinquefasciatus , 
Ae , aegypti and An, quadrimaculatus ; salient events in the midgut were 

specifically studied on a chronological basis, death of ingested 

h 
microfilariae being seen as early as 8 post-feed, leading Kartman 

(1953a) to conclude, "The present work has indicated that the fate of 

the microfilariae of D. .immitis in the mosquito midgut is undoubtedly 

a critical one for the completion of the parasite's life cycle. The 

preponderance of negative females in the Culex species is explained by 

the fact that the majority of the microfilariae are killed in the midguts 

during the first 24 after the infective meal" p. 69. Kartman (1953a) 

ultimately concluded that the differences might be due to the presence 

or absence of rapid bloodmeal coagulation, in turn tied to the secretion 

of salivary gland anticoagulant, which would increase the exposure time 

of microfilariae to antiblastic substances from the midgut or salivary 

gland3. In another study, the high resistance of Cx. nigripalpus to 

D. immitis infection was attributed to the entrapment of microfilariae 

in the rapidly-clotting bloodmeal and wounding of microfilariae, 

possibly from needle-sharp crystals of oxyhemaglobin, formed as the 

result of hemolysis of ingested red cells (Nayar and Sauerman, 1975)* 

As with Ox. quinquefasciatus in Kartman' 3 (l953 a ) study, the low 

intensity of infection for the group was attributable to the large 

portion of refractory (negative) mosquitoes. 

The gist of this body of research is that variation in susceptibility 
to filarial infections in most Cul ex and some Anopheles is a function of 
events first manifest in the midgut, which in most, but not all, mosquitoes 



35 

result in morbidity of most and sometimes all ingested microfilariae 
shortly after the mosquito feeds. These studies suggest an array 
of possible sources of the morbidity, including toxic factors, trauma 
or entrapment, or combinations of these. Furthermore, this variation 
occurs not only between species but within species; for example, 
Hu (1931) found 1 Cx. terr. it an 3 with largo numbers of prelarvae, 
although a vast majority of individuals in the 3pecie3 support only 
extremely low-level infections. Yen (1938), quoted above, alludes 
to this same phenomenon, while Kartman (1953 s ) states "the phenomenon 
of individual response to infection thus becomes of particular signifi- 
cance ..." and talks of "especially susceptible individuals" p. 66, and 
Nayar and Sauerman (1975) report their results in terms of proportions 
of the sample with a particular level of susceptibility. Thus, 
variation in susceptibility levels of individuals i3 itself a character- 
istic of the vector-parasite relationship in general, as is the normal 
development of filariae once the gut barrier is succesfully surmounted. 

Although microbes, including bacteria and riokettsia-type or- 
ganisms, have been observed in the bloodmeals of Gx. quinquefasciatus 
(Chao and Y/istreich, I960; Micks et al. , 1961), and that specific 
functions associated with digestion of the bloodmoal have been attributed 
to microbes (Arnal, 1950} Ferguson and Hicks, 196l), with alterations 
in the pattern of digestion resulting (Micks and Ferguson, 1961), no 
evidence has yet been found which associates microbes with filarial 
morbidity in any causal manner, although it is quite conceivable that 
occasionally microbes could cause a reduction in susceptibility by 
creating an unusually toxic environment in the midgut. However, the 
midgut in sugar-fed mosquitoes is never distended as during bloodfeeding, 



56 

storage of sugar being the function of the diverticulaej only small 
amounts of crop contents being passed to the midguts at any one time, 
and, thus, the volume of liquid medium is never very large. Terzian 
et al. (1952), and Terzian et al. (1953)» had reported alteration of 
normal susceptibility patterns in Ae. aegypti infected with Plasmodium 
gallinaceum , but they specifically attributed the effects to disturb- 
ances of the host physiochemical homeostasis, which produces the normal 
pattern. Thus, aside from what seem to be alterations of the suscepti- 
bility level of mosquitoes to Plasmodium induced by midgut environmental 
changes, with perhaps additional potential mechanisms from the absence 
of competition for nutrients, or absence of factors produced by the 
bacteria and used for host defenses (Micks and Ferguson, 196l), no 
antiblastic attribute has been assigned bacteria even in the case of 
Plasmodium , and it is unlikely that digestive enzymes, from either 
host or flora, which reach peak activity (Gooding, 1972) too late to 
be involved in the initial destruction of filarial parasites, are 
involved. Thus, the role of bacteria in mosquitoes, even as symbionts, 
is most likely associated with the crop in sugar-fed only mosquitoes, 
while with regard to the midgut bloodmeal, exert their influence only 
when enough time has passed to allow the population to build up. In 
fact, more than a third of An, crucians , 60% of An. quadrimaculatu3 and 
100/u of Ae. infirmatus examined for microbes were sterile (Hamilton, 
1975)» who also reported an absence of association of bacteria and early 
development of D. immitis in monocontaminated An . quadrimaculatus . 
Normal, albeit sluggish, microfilariae of D. immitia are seen in 
+24 bloodmeals in Ae. ae/m>ti , and it is questionable whether intact 
microfilariae are subject to enzymatic attack in mosquito midguts at 



57 

all (Kartman, 1953&)* Thus, the inherent inconsistency of microbial 
infection of mosquito midguts, compared with the consistency of the 
observed morbidity, does not support the contention that flora are 
involved in any significant way, even as sources of hemolysis (Ferguson 
and Kicks, I96I) or coagulins, in the determination of susceptibility 
to filarial infection. 

The vector-parasite relationship between Ae. aegypti and 
D. immitis is consistent only in the variability of the elements used 
in its characterization; this is a property of the Ae. aegypti t f ilariid 
relationships in general. The first of these elements is the decrease 
in survivorship of mosquitoes that ingest large numbers of micro- 
filariae,; first observed by Fulleborn (1908). Following this study, 
many reports using Ae. aegypti in conjunction with a variety of 
onchocercids, including Brugia (Wharton, 1957)* Dirofilaria tenuis 
(Pistey, 1958) and Wuchereria (Rosen, 1954) have alluded to this phenomenon, 
and specific studies have even focused on it (Kershaw et al. , 1953? Weiner 
and Bradley, 1970). Travis (1947) reported extremely high mortality rates 
for Ae. guamensis and Ae. pandani after ingestion of D. immitis ; nearly 
identical results were reported by Rosen (1954) for Ae. edgarl and Ae . 
polynensls infected with D. immitis on Tahiti. These studies support the 
view that failure to survive after ingesting even moderate levels of micro- 
filariae, while highly variable in expression, is actually one of the 
more consistent aspects of this vector-parasite relationship and serves 
as an excellent example of the reversibility of the epidemiological 
triad; not only does the vector affect parasite but the parasite can 
affect the vector, and this, in turn, can very significantly alter the 
vector potential of an infected population. Vector mortality ia not 



38 

specific to this mosquito — it occurs in several other mosquitoes in 
other genera (Weiner and Bradley, 1970; Hamilton and Bradley, 1979) — 
and it does not occur in all Aedes; for example, it is not a property 
of the vector-parasite relationship between D. immiti3 and Ae, triseriatu3 
(Intermill, 1973) or Ae. taeniorhynchus (H<iyar and Sauerman, 1975). 
The most frequent observation is that the midgut epithelium is being 
destroyed by the writhing and lashing of microfilariae, with rupturing 
of the cells and contamination of the hemocoel with gut contents, 
mortality from septicemia or toxemia ensuing. 

The second element used to characterize this relationship, first 
reported by Roubaud (1937), is the variation in susceptibility level 
to D. immitis infection between geographical strains of Ae. aegypti , 
as confirmed by Kartman (1953&)« Strain variation also characterizes 
the relationship between Ae. a.eg ypti and other onchocercids, such as 
B. malayi (MacDonald, 1962), and Wucher eria , Brugia and Dirofilaria 
(MacDonald and Ramachandran, 1965; Beckett and MacDonald, 1971; 
Obiamiwe, 1977) • A single, sex-linked, recessive gene, designated 
"r ", was found to control susceptibility level to Brugia and Wuchereria 
but not Dirofilaria (MacDonald, 1962; MacDonald and Ramachandran, 1965). 
Failure of penetration of the tubule cells in Ae. aegypti by D. immitis 
microfilariae was an expression of non-3usceptibility in one genetic 
study, which concluded that other factors, such an encapsulation, 
melanization and defecation, were not mechanisms mediating refractori- 
ness (McGreevy, 1972). 

The third element that can be used to characterize the host- 
parasite relationship between Ae. aegypti and p_. immi ti 3 is the arrest 



39 

of development of prelarvae after attainment of lodging in the target 
tissue, the Malpighian tubules, first described by Roubaud (1937) » 
although Hu (1931) alludes to this phenomenon briefly. Yen (1938) 
reported "retardation of growth" and presence of a prelarva in the 
Halpighian tubules on the tenth day post-infection in Ae. stimulans 
and Ae . canadensis , but normal development in most Ae. cinercus and 
Ae . vexans; it is of interest that this researcher observed the 
phenomenon in Culiseta inornata and Coquillettidia per turbans . It 
is not characteristic in Anopheles . Travis (1947) notes that in 
Ae . aegypti large numbers of larvae migrated to the Malpighian tubules 
from the stomach, and that in this species there was a much larger 
drop in the numbers that reached the sausage stage than in the other 
two species of Aedes , while Kartaan (1953a, p. 33) remarks "the Ae. aegypti 
females showed a predominance of unchanged microfilariae in their 
Malpighian tubules and this phenomenon was a daily finding throughout 
the experiment," which contrasted with his findings on the other 
mosquitoes investigated (An. quadrimaculatus and Cx. pi pi ens and 
Cx. quinquefasciatus ) ; further, "Some of the unchanged microfilariae 
in aegypti were still alive fifteen days after being ingested'.' p»33» 
observations corroborated by Nayar and Sauerman (1975)« 

The cause of this arrest has been speculated upon and there are 
several hypothesized mechanisms, each supported by their own evidence. 
While one of these, encapsulation, is often considered a separate 
element of the host-parasite relationship, probably because the 
phenomenon is so widely observed in mosquito-filariae relationships, 
there is no evidence, other than studies u3ing light microscopy 
observations, that definitely eliminates it from involvement. Pigmental 



40 



encapsulation of D. immitis occurred in Ae. vexans Malpighian tubules 
studied by Grassi and Noe (1900), and in all Aedes studied by Yen (1938), 
and he considered this to be the source of developmental arrest in Cu. 
inornata and Coo^. perturbans . Yen (1938) concluded that the capsules were 
the "shed skin" of the parasite "the products of degeneration," and that 
all capsules are within cells, and he disagreed with earlier reports 
alluding to a chitinous chemical nature in the capsules. Kartman 
(1953a) notes that "the invasion of Ae. aegypti by D. immitis was 
attended by a consistent encapsulation of the microfilariae, ..." p. 35. 
but only 12% of the mosquitoes dissected harbored these forms, and he 
concluded that the phenomenon was "... of little quantitative 
significance" p. 35. However, Lindemann (1977) describes lOQj 
encapsulation of microfilariae of D. immitis in the tubules of Ae. 
aegypti , although the infectious load was relatively light. 

Another possible mechanism is the laying down of membranes by 
the host around the body of the parasite. Membranes can play a very 
important role in host-parasite relationships involving intracellular 
parasites, with membranes of both host and parasite origin being laid 
down (Traeger, 1974); Ashley (1972) observed such membranes around 
D. immitis in Ae. aegypti tubules using electron microscopy, while 
more recent comparative studies along similar lines indicate that the 
phenomenon might be associated with developmental arrest (Bradley and 
Nayar, 1980). Since the parasite is predominantly intracellular in the 
early stages (Taylor, i960), relying on trans-cuticular absorbtion of 
nutrients (Nelson, 1964), the membranal isolation of the parasite offers 
an attractive hypothesis to explore. However, if the membranes are 
lysosomal, the failure of prelarvae to be digested and the presence of 



41 

living but moribund prelarvae up to 15 days post-feed must be ex- 
plained. Furthermore, as with encapsulation, the cause-effect aspect 
must be resolved, since enveloping of forms made moribund or killed 
by other mechanisms is quite possible, based on the current knowledge. 

Aside from physical isolation mechanisms, possible chemical 
mediation of arrest of development is indicated from the work of 
Weathersby and MoCall (1968) and Weathersby et al. , (1971) who found 
that substances in whole body extracts could alter susceptibility 
levels in Ae. ae^ypti to Plasmodium gallinaceum infections, factors that 
were apparently "antiblastic" in action. Atreptic (nutritional) sources 
of arrest are suggested in the work of Traeger (1974), who demonstrated 
a requirement by Plasmodium lophurae for adenosine triphosphate , but 
not as a purine, since the diphosphate form was not efficacious, but, 
more likely, as a co-factor in membrane transport. In the absence of 
the correct chemical substances, the prelarvae could be placed in a 
stress situation, continuing their lashing, thereby destroying the 
structure of the target tissue. 

Finally, the absence of requisite physical-chemical conditions 
in the tubules of Ae. aegypti might be the actual mechanism that causes 
the arrest; if the parasite requires a quiescent period prior to 
initiation of development, the disruption of the composition of the 
tubule cell3 by the filariae due to an inherent structural factor in 
these cell3 such as relatively fragile membranes, or the absence of 
proper turgor (osmotic pressure), could explain ultimate arrest. It 
is understandable that such microfilariae would be under a continual 
stress, unable to pass into the quiescent state. External physical 



42 

factors could affect development of the parasite, including temperature 
and pressure} humidity has been associated with varying levels of 
susceptibility (Basu and Rao, 1939)* 

The work of Roubaud (193?) suggests that the phenomenon of arrest 
of development is not universally distributed amongst the varying 
strains of Ae. aegypti , with susceptibility ranging from 100 percent 
to nearly total refractoriness, and it was logically the arrest of 
development that was involved in the genetic expression of decreased 
susceptibility, as suggested in the work of Kartman (l953 a ) and 
McGreevy (1972); MoGreevy et al. (1974). 

Thia historical review suggests that there is at least a general 
association between the type of mechanism that mediates susceptibility 
to filarial infections and the genus of the vector. The fate of the 
parasite can be decided at the gut level or target tissue level, as 
suggested by Nayar and Sauerman (1975) » but apparently not both in the 
same mosquito. Furthermore, the expression of these mechanisms is 
variable within species and, again, within strains. Regardless of 
site of action, the net result of both mechanisms is the reduction of 
host efficiency and, ultimately, vector potential. 



43 

Definitions and Usage 

In general, the terminology of Kartman (l953 a ) is employed here, 
with the following modifications and additions: 

Microfilariae (MF) — generally considered precocious embryoes 
because they lack the functional alimentary tract characteristic of 
other nematode first-stage larvae (Taylor, i960), in the present study, 
this is the form that occurs in association with vertebrate blood, which 
includes, therefore, both the vertebrate bloodstream and invertebrate 
gut. This form has no potential for development in either the vertebrate 
host blood or invertebrate alimentary canal. 

Prelarvae (PL) — the precocious embryoes, indistinguishable from 
microfilariae, but not found in association with the blood, having 
attained lodging in the target tissue, in the case of D. immiti3 , the 
Malpighian tubules. This form has the potential for development on the 
basis of environment. The terminology for microfilariae and prelarvae 
in the literature is inconsistent and confusing. To give just a few 
examples, Kartman (1953&) U3es the term microfilaria! to refer to all 
pre-developing stages, as does Taylor (i960), while Pistey (1958) 
assigns the term prelarvae once they have been ingested by the arthropod, 
which is closer to the usage here. Other criteria encountered were 
"G"-cell division, shortening, thickening; thus, the issue seems more 
one of semantics than biology. The present terminology is both logical 
and functional; logical, in that microfilariae have no potential to 
develop into larvae (no development of microfilariids while in associa- 
tion with the vertebrate blood has been reported) while prelarvae, 



having attained the invertebrate target tissue, do have that potential; 
a larva is a developing stage, with the prelarva its precursor; 
functional, in that each term is restrictive as to location, prelarvae 
never occurring in the gut. 

Developing Form (DF) — usually, as in Kartman ( 1953a)* any stage 
beyond prelarvae; in the present study, in the case of Culex nigripalpus , 
the prelarvae is equivalent to the developing form, since no barriers 
beyond this stage have been demonstrated in Culex tfilariid relation- 
ships; undeveloped prelarvae are not normally found at +120 in moderate 
infections. In Aedes ae.^ypti . this relationship does not hold; that is, 
prelarvae and developing forms are not equivalent, because undeveloped 
but living prelarvae are often found in the Malpighian tubules at +120* 
and even +240 . 

Susceptible (Mosquito) — a mosquito that supports the development of 
any filarial larvae (even one). This definition is far more inclusive 
than that of Hu (I93l)» where an individual was considered positive for 
susceptibility only when all the larvae seemed likely to complete 
development or already had; this usage would eliminate those mosquitoes 
harboring developing forms concurrently with undeveloped prelarvae; 
since these mosquitoes can become infective due to the former group, 
it is not logical to assign them a category that would imply that they 
are not susceptible. In the present case, the word "susceptible" is 
inclusive and does not designate a specific level of susceptibility, as 
"refractory" (see below) does. In this study, the word "susceptible" 
is used with or implies the presence of a qualifier for designation of 
a particular level, such as, "low-level," "moderate," " Anopheles 



45 

quadr imacul atus level," of susceptibility, and so on; thus, it is a 
relative term, covering an array of situations, the only thread 
connecting them being the presence of at least one developing form, 
regardless of the number ingested. 

Refractory (Mosquito) — a mosquito harboring no developing forms, 
regardless of number of microfilariae invested. 

Atreptic/Antiblastic (Weathersby and McCall, 1968) — terms used to 
designate the source of factors causing an event, the first referring to 
nutritional factors that operate by their absence ; that is, the absence 
of an essential factor, such a3 an amino acid or vitamin; the second 
refers to factors that operate by their presence , such as general and 
specific toxins. 



46 



Materials and Methods 

Entomological and Parasitologies! Material a and General Procedures 
Biological material 

The mixed breed, 18 kg male dog used in this study to both feed 
mosquitoes directly and to provide a source of microfilariae for other 
studies harbored a naturally-contracted heartworra load that produced a 

microfilaremia of 75-150 microfilariae/ul when sampled by venipuncture 

oo hr 
of the cephalic vein at approximately 10 + 1 . White leghorn chicks 

and chickens, aged from 2 weeks post-hatch to adults, were continuously 

available at the Florida Medical Entomology Laboratory (FMEL). 

Three mosquitoes, Cx. nigripalp us, Ae» aegypti and An. quadrimaculatua , 
each available as colony material courtesy of Br. J. K. Nayar of the 
Florida Medical Entomology Laboratory, Vero Beach, Florida, where these 
studies were conducted, were chosen, based on previous experimental 
use (Nayar and Sauerman, 1975) » to serve as models for particular 
susceptibility patterns, associated according to the literature with 
particular genera or portions of genera. The Cx. nigripalpus colony, 
established by J. S. Haager during 1975, possessed all the characteristics 
reported for the genus, and vas, furthermore, readily available year 
round in case of colony failure, The Vero Beach strain of Ae. aegypti, 
developed by J, K. Nayar during 1974, likewise produced arrest of develop- 
ment and exhibited variable susceptibility levels and vector mortality. 
The An, quadrimaculatus colony, originally from Gainesville, Florida, 
was essentially 100 percent comprised of susceptible individuals, and 
was, therefore, nonpareil as a microfilarial and prelarval vitality 
control. Rearing procedures for each species were conventional and are 



M 



described elsewhere (Nayar and Sauerman, 1975). Ox. nigrlpalpus 
used were between 7-21 days post-emergence, while An. quadrimacul atus 
and Ae. aegypti were between 7-14 days post-emergence, and all species 
were maintained in 15 cm holding cages on 10% sucrose solution at 
approximately 80ft relative humidity, until 24 prior to a scheduled 
feeding, when the sucrose was replaced with water. 

Feeding of mosquitoes 

For direct feeding on the dog, either the paw and lower leg were 
placed in the cage through the stockinet sock, or the dog's chest was 
laid down on top of the upward-facing screened side of the holding cage, 
while its head and rears were supported by the closed upper sides of 2 
other holding cages, padding added to make the animal as comfortable 
as possible to reduce struggling (Figure 11-1,2). In general the 
slowest-feeding of the 3 species, most Gx. nigripalpus fed within 30 
minutes; they are far more tenacious once they have initiated probing 
and are lees likely to be dislodged by momentary movements or leg 
twitches by the dog than the other 2 species, which, however, take their 
fill more rapidly. Furthermore, engorging by Cx. nigripalpus is delayed 
by the "pool-feeder" behavior, characteristic of many mosquitoes in the 
Culex genus, which leaves a small red dot approximately a mm in diameter, 
which take several days to disappear, as evidence of its visit; no such 
marks are produced by the feeding of the other 2 species. After re- 
moving and discarding unfed and partially-fod individuals, engorged 
specimens were removed to a 27 C bioroom where they were held until the 
particular experiment had them scheduled for dissection. 

For feeding mosquitoes via membrane, an apparatus was constructed 
which incorporated the improvements of V-'ade (1976) on the Rutledge e t al„ 



48 




Figure II-1 . Feeding methods: direct feeding of mosquitoes on 
legs of dog inserted through access sock. 




Figure II-2. Feeding methods: direct feeding of mosquitoes on 
shoulder of dog laid down on top of cage. 



49 

( 1964) » design with a direct-drive, variac-controlled stirring device, to 
prevent sedimentation, and temperature control of + 0.10 C (Figure II-3 
and 4)l as little as 0»25 ml of "blood could be used in the feeding 
tube, which was fashioned from a length of thick, boiler, glass 
tubing, and the entire waterbath-feeder tube-stirrer unit was sealed 
at the top after charging the feeder, which kept the relative humidity 
within the chamber near 100ft, reducing evaporation of blood from the 
tube. The entire feeding assembly was connected with tubing to a 
5-gallon tank containing a thermostatically controlled, 120-volt 
nichrome wire water heater and submersible, variac-controlled, "Little 
Giant" pump, along with the thermo-regulator and thermometer. Cx . 
nigripalpuB fed best on media warmed to approximately 34 C, while Ae. 
aegypti and An. quadrlmaculatus preferred 37 C. 

An array of membranes was subjected to preliminary testing for 
suitability for use as the feeder tube end-cover, including hog-gut 

sausage casings, Baudruche, chick skin, chicken or turkey crops, 

TM 

Parafilm, prophylactic contraceptive rubber, and Fourex natural 

animal membrane prophylactic contraceptives. While Ae. aegypti fed 

best on cleaned chick crops, which was also Rutl edge et__al. (1 964) best 

TM 
membrane, Cx. nigrlpalpus definitely preferred the Fourex (washed 

thoroughly before use to remove factory lubricants/preservatives), 

and this was fortunate, as each contraceptive yielded 8-10 high quality 

(stripped of all fascia and fiber) and durable membrane covers for the 

feeder, at approximately $0.10 per cover and far less time put into 

preparation than chick crops. An. quadrimacul atus were more catholic, 

although they showed a slight preference (preferences were not measured 

experimentally, but differences were quite obvious) for the higher 



50 




51 




Figure II-4. Feeding methods: membrane feeder in position on top 
of cage, with water bath reservoir (left) containing pump and 
thermoregulator. 



52 

feeding temperatures and chick crop membranes. Membranes were attached 
to the feeder tube with a rubber grommet, the outside diameter of which 
allowed a water tight fit to the feeder tube water bath, and 1-2 mm 
of tissue was allowed past the end of the tube, creating a bulging 
membrane just past the membrane-grommet interface. The adjustable 
stirrer was set about a mm above the membrane bottom, past the end of 
the feeder tube by a few mm, into the bulge caused by the weight of 
fluid. 

For blood feeding mosquitoes, 0.5 or 1 ml of blood (the amount 
depended on the number of mosquitoes to be fed) was drawn from a 
suitable leg vein on the dog (all 4 legs were used when schedules 
called for frequent feedings, but this did not amount to more than an 
ml per day under daily feeding schedules) and placed in a 10 x 75 mm 
test tube containing 0.1 ml saline (0.9O& NaCl, unless otherwise stated), 
for each ml blood and anticoagulant, unless glass bead-defibrinated 
blood was used, in which case the 0.1 ml of saline only was used. 
Three anticoagulants were used: 1-2 mg/ml blood sodium heparin, with 
an activity of 135 units/mg (Nutritional Biocheraicals Co.) (the minimum 
amount used is more than 10X the amount required to prevent the blood 
from clotting); 3.8 rag sodium oitrate (0.387o)/ral blood; 2 mg EDTA 
( ethyl enediamine tetraacetic acid)/ml blood. For glass bead-defibrinating, 
the freshly drawn blood was placed in a 25 ml flask half-filled with 3 "an 
glass beads (Sigma Chemical Co.) and with 0.1 ml saline; the stoppered 
flask was attached to a rotator and the preparation churned for 30 
minutes at 40-50 revolutions/minute; anticoagulant-treated blood was 
either rotated or shaken every few minutes after more vigorous initial 
shaking to insure distribution of anticoagulant; additives were mixed 



53 

with the blood simultaneously with anticoagulant. Chicken blood 
drawn from the wing vein and jird blood from cardiac puncture were 
processed similarly. Heparinized plasma was obtained using conventional 
and centrifugation techniques; serum was obtained by placing a 5 ml 
aliquot of freshly drawn blood into a Sigma Thrombin Vial (stock 
number 850-1), mixing and incubating the vial for 2 at 37 C. This 
technique is more likely to convert all fibrinogen present to fibrin 
than conventional techniques, which can allow fibrinogen to be present 
after clotting since the plasma thrombin levels are variable. Washed 
red cells were prepared by 3X washing in saline after initial centrifuga- 
tion of anticoagulant-treated whole blood and decanting of plasma. 

The fluids were added to the feeder tube via Pasteur pipette. The 
feeder tube with membrane in place is maintained until this moment with 
a few ml of saline in the tube and the membrane suspended by the grommet 
in a saline—filled small dish; the saline is discarded and the tube 
and inner membrane surface are dried with a length of rolled paper 
towel, dabbing the membrane gently. After securing the feeder tube in 
the bath and setting the stirrer length, the tube is brought to tempera- 
ture via the water bath, the fluid added, and the apparatus placed on 
top of the cage of mosquitoes, the membrane-covered tip of the feeder 
tube barely inserted into the sugar vial hole. The stirrer is quickly 
placed back down into the feeder tube, and the entire system sealed with 
appropriately-sized plastic lids. The stirrer speed can be controlled, 
but 100-200 revolutions per minute was the usual setting. No effect on 
microfilariae from the paddle action was observed, and the results of 
experiments are not indicative of vitality loss from this source. 



54 

Separation of microfilariae 

Microfilariae free from the blood were needed for several pro- 
cedures, and these were obtained by placing an aliquot of freshly 
drawn anticoagulant-treated blood into a 15 ml centrifuge tube, 
adding citrated (0.3/0 saline up to 15 ml, mixing thoroughly, adding 
l(T/o saponin drop by drop — 3-4 drops usually sufficing for less than 
an ml of blood — and checking for the typical clearing of the mixture 
after the addition and mixing of each drop-. As soon as clearing is 
noted, the mixture is immediately centrifuged at 900 rpm for 5-6 
minutes, the clear red fluid decanted and replaced with fresh, non- 
citrated normal saline, the pellet agitated into the saline, and the 
procedure repeated once more. The pellet of microfilariae is very soft 
due to the relatively slow separation speed, and decanting was achieved 
using a rubber bulb-type automatic pipetter on a 25 ml volumetric 
pipette. If these microfilariae were to be suspended in another fluid, 
the fluid was simply added to the final pellet and gently vortexed; 
otherwise, if the microfilariae were to be used in saline, a few drops 
of the saline was added into the centrifuge tube, vortexed gently and, 
prior to use, transferred as a 100-200 ul drop to a siliconized slide, 
then covered with a high humidity cap. Microfilariae in such drops 
tend to aggregate in the center of the drop, facilitating collecting in 
a concentrated form with minimal dilution from the saline, an important 
consideration in certain procedures, such as testing of extracts. When 
a procedure necessitated that microfilariae remain in non-blood fraction 
fluids such as saline for more than 1 , the final washing and suspension 
was done using Hank's or Locke 1 3 buffered salines. High humidity caps, 
to curtail dessication of droplets, vere fashioned from 3 ml-syringe 



55 

sterile container end caps which were atomized with saline solution 
just prior to use; condensation droplets usually formed on the dry 
area of the slide surrounding the drop under the cap within 30 seconds, 
and continued vitality after several hours under such conditions 
suggests high efficacy in the system, and droplets as small as 20 pi 
could be maintained thusly. 

Dissection for midgut microfilariae and tubule prelarvae 

Conventional technique was used to remove insect midguts and 
Malpighian tubules, the salient aspects of which will be described. 
The materials of choice are 2 pairs of insect forceps, insect or 
normal saline, siliconized microscope slides with 24 x 50 mm cover- 
slips, and a 10-20X dissecting scope with accessories. With the 
specimen ventral side up and the tip of the abdomen in a drop of 
saline, the thorax is grasped with one forceps and subterminal segment 
with the other, which is used to pull the hindgut and rectum into the 
droplet. If the head is not removed, the gut usually disconnects at 
the cardiac sphincter yielding the midgut, tubules and hindgut, while 
sometimes only the hindgut and tubules are obtained when the breakage 
occurs at the pyloris. With the head removed, with practice and if 
care is taken, the 3 diverticulae are also obtained, the breakage 
usually occurring at the esophagus, a short tube connecting the posterior 
of the pharyngeal pump with the proventriculus. If the midgut is grasped 
at the pyloris, and terminal or subterminal segments grasped and pulled 
away, the disconnection usually yields the midgut and tubules, which are 
easily separated by grasping the anterior of the midgut and pulling 
the tubules away, grasping them as proximal to the gut as possible. 
Although tubules can be examined individually on slides, each set with 



56 

its own coverslip, as many as 15 sets could be processed on the same 
slide by simply placing each set in its own 10 ^ul droplet (the initial 
droplets can be far larger to avoid problems caused by dessication; 
but using lung-powered drawn capillaries in mouth aspirators, the 
final volume just prior to coverslipping is quickly reduced to ap- 
proximately 10 ul) and a 24 x 50 or 24 x 60 mm coverslip evenly 
lowered onto the droplets. It is important not to have too much 
liquid under the slip, since compression squeezes out contents from 
under the slip and material can be lost, or preparations mixed, when the 
pressure is released. In the non-compressed tubule, prelarvae are 
usually quite occult, but compression and release disintegrates the 
tubules, releasing the prelarvae into the medium in the vicinity of 
the tubule debris. Unless so stated, all prelarvae in each tubule 
were searched for and counted. Unlike sausages and later stages, the 
prelarva is a durable creature and usually quite vital following 
compression, thrasing and undulating in normal fashion (Figures 11-5,6). 

Host efficiency assay 

To determine the number of microfilariae ingested, the epithelium 
is gently removed from around the bloodmeal and shaken to dislodge 
parts of meals; this is a simple matter in Anopheles and Qui ex where 
the meal is rapidly isolated by the peritrophic membrane, more difficult 
with Ae. aegypti , in which no membrane forms for many hours, and. 
portions of the bloodmeal are embedded in crypts in the epithelium. 
However, unless steps are taken to prevent it, the bloodmeal coagulates 
rapidly in Gx. nigripalpas (Nayar and Sauerman, 1975), trapping many 
microfilariae in the rubbery clot, in which visualization of the micro- 
filariae is impeded. Again, as with prelarvae, the clot must be subjected 



57 




Figure II-5. Malpighian tubule squash for prelarval count; 
in vitro bright field; x40. 




Figure II-6. Malpighian tubule squash for sausages; in vitro ; 
Nomarski optical systems; x100. 



58 



to compression to reveal the presence of the larvae or to release the 
microfilariae into the medium, a compression of far greater force than 
used for tubules, and even with this, the genuine clot is not dissembled, 
but merely springs back into shape; it is this characteristic which 
serves as proof of coagulation occurrence, since agglutinated meals, 
after subjected to compression, do not re-coalesce but remain dissipated. 
Interference with counting due to coagulation is not a problem in the 
other species, if dissections are performed soon after feeding, and, 
even if they are not, the number of trapped microfilariae is far less 
than in Culex , where the problem can be eliminated only by feeding with 
glass bead-defibrinated blood or microfilariae suspended in serum. 
Interference with counting from red cells is eliminated by the addition 
of a minute amount of (0.1 - 0.5^1 ) 10/i, saponin into the droplet of 
saline containing the meal, swirling with a lung-powered air jet through 
a drawn capillary until the characteristic transparent red color of 
hemolyzed red cells is seen. Using the same capillary used for swirling, 
the preparation is transferred to a modified Biles (1973) hemacytometer 
type counting chamber, which was found after comparative testing to 
out-perform the next best vital counting device, the Sedgewiok-Rafter 
chamber, by a wide margin, and this device was used in connection with 
a variety of procedures in this study. The only modification from 
Biles (1973) was the use of No. 2 coverslip pieces as supports for 
examining very small quantities of fluid (less than 1 pi). The compres- 
sion produced a flattened disc at least a few mm in diameter. For 
larger volumes, the design was essentially that of Biles (1973)« Not 
only are there no obscuring edges in such a chamber, there is no tendency 
for the liquid to travel (chambers must be siliconized to work efficiently)^ 



59 

and evaporation is minimal. One bloodmeal in aaline could be separated 
out into 100 2 ul droplets in the chamber and scanned efficiently and 
quickly. Some losses due to adherence of microfilariae to the sides 
of capillaries, which happens even when they are siliconized, undoubted- 
ly occurs, but this is not significant when the microfilaremia is as 
high as it was in this study. 

The foregoing procedures generate several data types, including 
number of microfilariae ingested per mosquito and average number of 
microfilariae per mosquito per experimental group, number of prelarvae 
per mosquito and average number of prelarvae per mosquito in a group, 
proportion of prelarvae of total ingested microfilariae, called the 
"host efficiency" index by its inventor, Kartman (1953a), and the 
frequency distribution of prelarvae per mosquito in a group, which 
can be used to form a "susceptibility" profile based on a comparison 
with the susceptible model, An. quadrimaculatus . The advantage of the 
prelarvae count is that it accurately measures the resultant of forces 
manifested in the midgut, while avoiding difficulties and added variables 
encountered with older infections. It is a characteristic of the Culex 
genus in its relationship with D. immitis that once the gut barriers 
are successfully surmounted, and prelarvae are safely in the tubules, 
development proceeds from that point without significant impediment; 
thus, with Culex , the prelarval count is a good estimator of vector 
potential, 3ince the developing form rates, used here to demonstrate 
barriers past the midgut, was essentially the same as in the susceptible 
model (the test does not discriminate amongst sources of poor host 
efficiency, this being the purpose of the vitality assay, to be 
described in a later section). Thus, it is this assay that produces 



60 



the frequency distribution of susceptibilities by which the species 
is characterized. The most frequently occurring values of prelarvae 
per mosquito for Cx. quinqu ef asc i atus (Kartman 1953a) and Cx. nlgripalpus 
(Nayar and Sauerman, 1975) are low to negative, and thus the mosquito 
as a species is placed in a low susceptibility category regarding its 
veotor-parasita relationship with D. immitis , while the opposite is 
true for An. quadrimacul atu3 . Further barriers beyond the gut are 
characteristic of the Ae. aegypti iD. immitis vector-parasite relation- 
sMp, and, therefore, this assay is inappropriate for measuring 
susceptibility in that species. With Ae. aegypti , alterations in 
susceptibility level are detected using the developing form assay, in 

which Malpighian tubules are removed and inspected for the presence 

h 

and portion of sausage forms at 120 + 24 after feeding. As in the 

prelarvae per female assay for Qui ex , sets of tubules are subjected to 
coverslip pressure; if considerable pressure is required to reveal all 
forms, this cannot be discontinued until the count is oomplete, as 
upon release, the soft-bodied sausages are no longer recognizable. 
The allowable interval for dissection was from 96 to 144 after 
feeding, but while later times could probably be used without loss of 
information, efforts were always directed, in the present study, towards 
conducting dissections at +120 . Under normal experimental conditions, 
most D. immitis will have "shortened" to sausages by +72 (Kartman, 
1953a; Nayar and Sauerman, 1975) » and preliminary studies indicated, 
as HcGreevy (1972) reported, that most prelarvae that fail to transform 
to sausages by 48-72 are either moribund or dead. There is never any 
sign of bacterial activity in the tubules, which are presumed to be 
sterile, since dead prelarvae, while sometimes encapsulated, are not 
digested by microbial activity. 



61 



Microfilaria! (and prelarval) vitality 

Normal microfilariae (includes prelarvae for purposes of this 
discussion) appear as a vigorously thrashing and rhythmically un- 
dulating organisms, the serpentine movements occurring concurrently 
with the twisting and turning back on itself. This is also character- 
istic of prelarvae up to about 48 after attaining the tubule environ- 
ment. Two type3 of morphological appearance for morbid microfilariae — ■ 
dead and intact and dead and disintegrating — were described by Kartman 
(1953a) as part of an assay. In this study, all immobile forms are 
aggregated with those exhibiting bizarre movements into a single 
category of "moribund," or dying, microfilariae; not included in this 
category are sluggishly-moving but rhythmically undulating individuals, 
a situation that most frequently occurs after they have remained in 
the particular medium for at least 30 minutes and is evidence of a 
non-supportive, but not necessarily toxic, environment where morbidity 
is a function of the absence of required (atreptic) factors in proper 
balance. On the other hand, if morbidity was induced within 10 minutes, 
this was taken as a sign of the presence of antlblastic toxins, even if 
normal but rapidly damping down undulations accompanied the loss of 
vigor. Any interference at all with rhythmic undulations was interpreted 
as the effect of physical or chemical trauma, sinoe preliminary observa- 
tions indicated that such larvae do not recover. Although there is 
good agreement in the literature that total vacuolization of the micro- 
filariae is synonomous with death, such larvae were still placed in the 
more inclusive moribund category. The assay generates several data 
types, including number of microfilariae or prelarvae that are morbid 
in an individual mosquito, average number of moribund microfilariae in 



62 

a mosquito in a group of mosquitoes, and a vitality profile, since 
vitality is not an all-or-nothing occurrence in the individual but 
instead is a matter of proportions of moribund microfilariae in an 
individual mosquito, which generates a frequency distribution for a 
particular group, allowing a comparison of distributions without 
losing sight of individuals. This is an all-important consideration 
in the present study, since it is often the small portion of individuals 
exhibiting behavior uncharacteristic in terms of the rest of the group, 
to a point where it is clear that they represent a different treatment, 
which are most illuminating in investigating the problem. The Biles 
(1973) hemacytometer was used for all determinations, since this method 
reduces factors that could affect vitality due to atreptic deterioration 
of the microenvironment. The only exception was direct ooverslip 
compression preparation of extracts, to be described below, in which 
no transfers were made. 

Coagulation time 

Conventional techniques were utilized to measure coagulation time; 
a fibrin rake with tines approximately 1 mm apart was fashioned using 
minuten pins epoxied to a wood applicator stick. Detection of first 
threads, which event serves as the basis for some automatio fibrinometer- 
type devices, were easily detected, and the growth of the clot could be 
monitored, which was necessary, since in cases where the amount anti- 
coagulant is small, clotting can cease, making questionable its equati- 
bility with fully clotted material. A high level of accuracy was not 
necessary for this study and accuracy decreases with the number of 
samples being simultaneously tested, if the number of operators remains 
constant. For rapidly clotting preparations, fewer samples were monitored, 



63 



if accuracy was desired. The maximum number sampled at any one time 
in this study was 6, 3 of which were controls, since controls were run 
on the same slide as the experimental group in the event that impurities 
on the glassware influence results, .tiach sample on a siliconized slide 
was covered after raking, which consumed about 3 seconds, with a high 
humidity cap, constructed as described previously in this section. 

Hemolysis 

Chicken erythrocytes are nucleated, and when hemolysis of the cell 
occurs, as with distilled water or saponin, the free nuclei liberated 
into the medium serve as intact markers of the event. On Giemsa- 
stained thin smears, solitary nuclei, which disintegrate as they dry 
on the slide into an amorphorous round mass often with feathered edges, 
stain the expected intense blue while nuclei in intact cells are oval 
and surrounded by the pinkish-orange cytoplasm. Under certain conditions, 
free nuclei tend to aggregate; such aggregation is reduced when a 

o R 

minute amount of Triton-X 100 or Tween-80 i3 added, at a rate of 
approximately 10-50 jjl/liter saline. In the Biles (1973) hemacytometer, 
nuclei present themselves as totally spherical particles measuring 
approximately 2 u in diameter. Both nuclei and intact cells sink to 
the floor of the hemacytometer a few minutes after preparation. The 
advantage of this method of determining hemolysis over conventional 
photometric techniques is far greater simplicity and not having to be 
concerned with an inherent contributor to the total variation caused by 
fluctuations in bloodmeal size. Preliminary experimentation indicated 
that hemolysis was variable from individual to individual, and thus 
plotting of hemolysis rates of individuals would generate a frequency 
distribution that could be analyzed and used in comparing treatments by 



64 



setting up profiles, where the bizarre elements are not lost in the 
average. Furthermore, if hemolysis in extracts was measured photo- 
metrically the small aliquots and low hemolysis rates would create 
more technical difficulties, while if dog or human erythrocytes are 
used, the exact number introduced would have to be determined; with 
chick cells, one can sample from a large distribution of cells, as 
in a clinical white cell differential count, or merely count all cells 
and nuclei. 

Extract preparation 

Extracts of salivary glands (SG) and midguts (MG) were prepared 
following the method of Day (1954)» with some modifications to be 
described. Salivary gland extract (SGE) was prepared by removing 
pairs of salivary glands into saline in a high humidity chamber until 
the required number wa3 accumulated, when the saline was replaced with 
distilled water; the rapid movement of fluid and particles (spherules) 
from the gland cells into the saline wa3 immediately evident, as described 
by Day (1954). The first modification came with removal and holding 
of this first extraction in a capillary, leaving only the SG, which are 
subjected to coverslip squashing with insect forceps under the dissecting 
scope; all cells are totally disintegrated, an edge of coverslip lifted 
a mm from the slide and the original fluid re-introduced and mixed with 
the squashed material and fluid. The slip is removed and the prepara- 
tion placed at 4 G in a frost-free refrigerator, which dissicates the 
preparation within a few hours, while inhibiting any microbial growth. 
Just prior to use, the slide with extract is placed in a high humidity 
chamber and saline added in the amount called for in the test protocol. 



65 

When KG are placed in distilled water, they swell within a 
minute, and the epithelium peels away from the basement membrane as it 
disintegrates, yielding a milky suspension inside the clear membrane. 
Such sacs are sucked through the orifice of a capillary drawn to a size 
that requires squeezing of the sac to get through it. After drawing 
the sacs through the orifice three times, totally macerating the 
material, the preparation is filtered by sucking it through a sterile 
cotton plug capillary filter with 2, 10 pi distilled water washes, 
yielding a clear fluid, which is henceforth processed as was SGE. 
In addition to extracts of these organs, fresh coverslip squashes of 
SG and KG were prepared to which were added microfilariae or chick red 
cells, to support the results of extracts and to provide factors that 
might be destroyed by extract preparation. 

One other substance prepared for testing its effects on micro- 
filaria! vitality and hemolysis was blood meal extract, which was the 
liquid portion of centrifuged blood meal3. Ten fresh blood meals were 
placed together in a drop of saline which was then drained; the meals 
were teased apart after the epithelium was removed, and meals were 
pushed down the cut end of a Pasteur pipette, the end of which was 
sealed with heat fusion. Making the cut where the glass began its 
flare facilitated the process, since this created a small funnel which 
helped to get the material into the tube without loss. These were then 
centrifuged at high speed for 15 minutes; the solid material was dis- 
carded after separating the tube at the junction of liquid and solid 
using a diamond pencil. 



66 

Additional Procedures and Materials 

Inoculation per ano , also known as the "enema" technique, of Cx. 
niffripalpus and other species with microfilariae and blood components 
was accomplished using drawn capillaries, the ends of which were fire- 
polished to facilitate penetration while avoiding rupture of the gut. 
Tips were brought close to the flame of an alcohol lamp from below, 
and, depending on the fineness of the draw, held a few mm from the flame 
for a moment and examined for polishing under the scope, A brief 
flicker of yellow color in the flame signaled that polishing had 
commenced, but complete sealing can easily occur, in which case the 
effort is simply renewed, until the proper result is attained. As 
much as 4 ul of fluid could be introduced in this manner, although 
2-3 ul was the amount most often delivered. Mosquitoes were prepared 
for per ano procedure by attaching them with white glue to a slide by 
the anterior thorax; the slide was inverted and placed at an angle so 
that upon drying and re-inversion the tip of the abdomen projected 
upward and slightly toward the operator. A scalpel edge applied to the 
glued area easily dislodges treated mosquitoes into appropriate holding 
containers. 

Injection of the midgut blood meal with microfilariae was easily 
accomplished using drawn capillaries; an abrupt draw gives the shaft 
stability, as flexion is undesirable. As with clinical technique, the 
best sign that the inoculating shaft is in proper position is the appear- 
ance of cells in the shaft extremity; the outward pressure exerted by 
the meal is usually sufficient for this to occur, although very light 
suction is sometimes helpful. Before inoculating the fluid, a brief 
moment is allowed to pass to permit hemolymph-sealing of the area. 



67 

Gradual delivery curtails the tendency of gut contents to leak when 
pressure builds. Forceps tines applied next to each side of the 
capillary shaft near the point of penetration reduces leakage after 
withdrawal. Injection capillaries are more finely drawn and sharply 
pointed than per ano capillaries. 

Excised midguts were inoculated with microfilariae and blood 
fractions at the cardiac sphincter, just posterior and adjacent to the 
origins of the diverticulae. The needle for such a procedure is fire- 
polished, as for enema, to facilitate injury-free penetration, which is 
accomplished by grasping the edge of the sphincter with a forceps, and 
inserting the needle through 1he sphincter down into the anterior midgut, 
all the way into the far more expanded area of the posterior midgut, 
which contains the secretory epithelium. 

Preconditioning 

Buffers, anticoagulants and antibiotics were used in specific 
experiments to condition mosquitoes prior to and during feeding} the 
buffers and anticoagulants are described in the Results section. Anti- 
biotics mixtures, in sugar or saline, depending on the experiment, were 
prepared as follows: 50 ug each of neomycin sulphate (675 mcg/mg), 
streptomycin sulphate, sodium penicillin G, and 100 p.g nystatin (anti- 
fungal antibiotic) per 100 ml saline or sucrose for preconditioning, or 
for feeding antibiotic simultaneously with blood, 500 jig of each anti- 
biotic is then mixed with 1 ml of the blood. 

Per Os bloodf ceding via sugar-feeding mode 

The feeding apparatus for this procedure is shown in Iigure II-7. 
Several small 6 mm O.D. glass tubes each about 3 cm long with a 4 mm 



68 



3 •5mm 



■-■7: 



STOPPERS 



POSITIONING 
GROMMET 



FEEDER TUBES 



FEEDER TUBE 
HOLDER AND 
COVER PLATS 




V 



/ 



MOSQUITO 

HOLDING 

GUP 



Figure II-7, Special apparatus for blood feeding mosquitoes in 
the sugar feeding mode-schematic. 



69 

inner diameter were charged with sugar solution containing microfilariae 
or red cells. The end of the tube that projected into the cup was 
ground, to allow even dispersion of the substance around the orifice; 
about 100 )xl could be placed in such tubes without affecting the important 
operating characteristic, namely, the maintenance of a non-protruding 
column of liquid in the tube. The purpose of this is to prevent the 
mosquito from drinking in areas that are free of microfilariae or 
cells; these settle to the bottom by gravity, and, if a bulge occurs, 
clear areas around the upper edges of the protrusion can allow particle- 
free imbibition. All such preparations are offered at room temperature 
only. Mosquitoes to be so fed are starved for at least 24 before 
feeding. The top of each tube projecting from the tube holder on top 
of the cup is sealed with a cork to retard evaporation. 

Wound Assay 

The presence of injured microfilariae in the midguts of Cx . 
nigrlpalpus was first reported by Nayar and Sauerman, (1975)> although 
the same condition was reported previously in other Culex and some 
Anopheles by Goluzzi and Trabucchi (1968), using Dirofilaria repens , 
and in Cx. t erritans - ingested Foleyella flexicauda (Benach and Crans, 
1975). In the present study, microfilariae were separated from the 
other blood meal components after hemolysis of red cells to facilitate 
inspection, placed in droplets on a slide and allowed to dry. The 
method of Awogun (1978) was used, with the modification that preliminary 
fixing with acetic is not necessary, the addition of the Hoyer-fixative- 
stain (hematoxylin) mixture serving the purpose quite adequately. The 
great advantage of this technique is the simultaneous fixation, staining 
and mounting, and the staining results are excellent for the purpose. 



70 

When large numbers of microfilariae were present in the preparation, 
a sample count was taken to determine the occurrence and rate of 
wounding, which, like vitality and hemolysis, was variable in Cx. 
nigripalpus , and thus a group of mosquitoes generated a frequency 
distribution of portion wounded. 

Histological Examinations 

Material for histological examination was prepared essentially as 
described in Nayar and Sauerman (1975)» Serial sections of "J yx. slices 
of groups undergoing a variety of treatments were prepared and examined 
for differences between them and controls or other model mosquitoes, 
regarding blood meal processing and other features that could help to 
explain the vector-parasite relationships. 

Smears 

Thick smears of midguts were heat-fixed and stained in Loeffler's 
methylene blue for bacteriological examination. While midguts were 
usually devoid of bacteria or supported barely detectable populations, 
in some cases large discrete boluses of bacteria were present, apparently 
digesting the meconium. Antibiotic preconditioning was apparently 
successful in destroying these organisms, if the smears can be taken as 
a measure of activity (Micks and Ferguson, 196l), but culturing of 
midguts was not done; thus, although digestive processes were disturbed 
by administration of the antibiotic, aa to whsther it really eliminated 
the flora was not determined. It seems safer to say that "suppression" 
occurred. 

Thin smears of blood and other substances were usually fixed with 
absolute methanol and stained with Giemsa 1:10 for 10 minutes and washed 



71 

in pH 7-5 phosphate buffer. This was adequate, although superior 
results are obtained if the smear is stained for J>0 minutes. 

Data Presentation and Statistical Analysis 

Counts of microfilariae, prelarvae, hemolyzed cells and vitality 
levels from individual mosquitoes are highly variable and therefore 
generate frequency distributions, when groups of such individuals are 
plotted. In several instances, it was apparent that these distribu- 
tions were not "normal" and that simple presentation of arithmetic means 
and standard deviations would be inappropriate in terms of representing 
central tendency and detecting shifts in individual composition of 
particular regions of the distribution, and, therefore, two representa- 
tions of the data are presented. One is the geometric mean, the anti- 
logarithm of the arithmetic mean of log values of elemental data, and 
geometric standard deviation. The basis of the normal standard devia- 
tion is the squaring of deviant values, thereby conferring greater 
weight on deviant values, and, since the average deviation decreases 
with increases in sample size, logically places weight on sample size. 
But, when data is heavily "skewed," as was at times the present case 
due to large portions of and low value counts, the few high values 
pull the arithmetic mean disproportionately away from the "average" or 
"representative" mosquito, the arithmetic mean being the most efficient 
locator of central tendency when the distribution i3 normal (Langley, 
1971 )• Use of the geometric mean, since it has it3 basis in logarithmic 
transformation, reduces the weight of the extreme values and is more 
representative of central tendency, which might otherwise be better 
located by the median. 



72 

The geometric mean, when used with heavily skewed data, also, as 
the above might imply, transforms the shape of the distribution from 
skewed to normal, thereby fulfilling a requirement of parametric statis- 
tics (normal -shaped distribution), thereby allowing the use of this 
more powerful body of statistics. These methods of hypothesis testing 
cannot be used with data that are heavily skewed or in any way non- 
normal, and thus non-parametric methods, which are less efficient 
(Sokal and Rohlf, 1969 ), would have been mandated without further 
transformation. The parametric statistic used for hypothesis testing 
in this study was the Student's 't' distribution, calculated on the 
basis of different sample sizes (Steel and Torrie, 196O; Snedecor and 
Cochran, 1968), where the sums of squared deviations are 'pooled* and 
divided by the sum of sample N's - 2, forming a variance that is used 
to calculate 't' by dividing the difference between means by the sample 
estimate of the standard error of that difference (calculated from the 
pooled variance). Degrees of freedom are calculated from the combined 
sample sizes - 2. 

The second representation of data is the "profile," which is 
essentially the grouping of a frequency distribution into classes allows 
a more meaningful visualization of shifts or dislocations possibly 
associated with a treatment than ungrouped basic data. The establishment 
of categories is arbitrary, as it is in all such cases, and each category 
is not of the same proportional size in all profiles, since special 
attention to the more deviant classes was of paramount importance re- 
garding investigation of mechanisms. Mean3 and standard deviations, 
while excellent statistics for central tendency and dispersion, lost 
sight of individuals and their position on a measurement continuum, and 



7 5 

different distributiona can have the same means and same average squared 
deviation. The profile, the categories of which are arbitrarily assigned 
equivalent levels of susceptibility, allows visualization of the shifts 
of individuals through categories associated with different treatments 
without obfuscation from basic data clutter. 

The relationship between number of microfilariae ingested and 
number of prelarvae in the tubules was statistically analyzed using 
conventional "least-squares" formulae. Correlation coefficients are 
included as a measure of how well the linear regression line approximates 
the data. 

The following were used in calculations: 

Geometric meaji (X) and standard deviation (s) 

X geometric = antilog (log £ X/N) - 1 

2 2 

s geometric = antilog Z log X - (E log X) 

N 



N-l 



t = V X 2 
s - - 
*1- X 2 




N ♦ N - 2** 



(t-test statistic) (sample estimate of (pooled variance) 
standard error of 
difference) 



* Each x value was incremented by 1 prior to calculation of mean and 
standard deviation of logarithmic values because of the presence the 
values in the data. Therefore, the geometric values are actually 
the antilog - 1 of the mean and standard deviation. 

** Degrees of freedom 



74 
Linear regression and correlation coefficient 



slope = t XY - I X % Y 

N 



IX 2 - (IX) 2 
N 

Y-intercept = IY - (slope) ( gx) 

N 

correlation coefficient = JXY / /(tX 2 )(tY 2 ) 
Experimental Results and Discussion 

Culex Susceptibility Patterns - Background 

To investigate the mechanisms that would explain susceptibility 
level variation of the pattern found in Qui ex spp., Gx. nigripalpus 
was chosen because the salient characteristics are more sharply defined 
in this species than in Cx. quinquefasciatus (Nayar and Sauerman, 1975) » 
with greater portions of mosquitoes exhibiting the characteristic 
immobilizing of ingested microfilariae and resultant low intensity of 
infection} the additional potential mechanisms of rapid coagulation of 
the bloodmeal and hemolysis of red cells with crystal formation are 
likewise more extreme than in the other Culex described in the literature 
with the possible exception of Cx. territans , all of which implies that 
experimentally-induced dislocations will be more rapidly and accurately 
detected. Furthermore, this insect is readily available year round in 
the region in which this study was conducted and has been implicated in 
the transmission of St. Louis encephalitis, turkey malaria and dog 
heartworm (Nayar, 1980). It is the aim of thi3 study to explore factors 
that induce entrapment and loss of microfilarial vitality utilizing and 
extending the addition: deletion strategy of Kartman (I955a)> who essentially 



75 



was adding the "missing" salivary gland anticoagulant. Entrapment is 
visualized as a function of absence of anticoagulant secretion by the 
salivary glands, as first suggested by Kartman ( 1955a). or the presence 
of midgut coagulants, which have been described in mosquitoes (Buck, 
1937). Miorofilarial vitality loss presents a more complex problem, 
with possible sources including the presence of antiblastic, toxic 
factors from host or host-flora (which may indirectly depend on movement 
hindrance for their effect), and the traumatizing of microfilariae by 
pharyngeal valve spines or needle-sharp oxyhemaglobin crystals, them- 
selves the product of hemolysis due to host factors or flora metobolites. 

The Effects of Hindrance of Migration of Microfilariae to the Malpighian 
Tubul es 

Introduction 

Although the increase in migration rate of D. lmmitlg microfilariae 
to the Malpighian tubules in Ae. aegypti , blood fed via membrane on 
heparinized, infectious dog blood, was not ultimately linked to a change 
in either "host efficiency" or "infective potential," the significant 
fact remains that Kartman (1953a) had succeeded in experimentally 
altering at least one phase of the Ae. aeflyptiiD. immitis vector-parasite 
relationship. The experiment was designed to test a hypothesis, based 
on his observation that the coagulation time of the ingested bloodmeal 
is a salient variable distinguishing the highly susceptible An. 
quadrimaculatus from the far less susceptible Ae. aegy pti and Cx. 
q uinouefasciatus , that the speed of migration, or conversely, the 
hindrance of movement, of ingested microfilariae through the midgut to 
the target tissue determined the susceptibility level, possibly by in- 
creasing or decreasing the exposure time to salivary gland or midgut 



76 

secretions that were antiblastic with regard to the microfilariae. The 
ingenius tactic of "adding" an absent factor, in this case, salivary- 
gland anticoagulant, to simulate the susceptible mosquito, to determine 
if the variable factor mediated the susceptibility level, was a hallmark 
in the investigation of filarial host-paraoite relationships. 

However, although Kartman (1553a) bad also determined that the 
pattern of infection in his Cul ex group 3trongly suggested digestive 
process involvement, he had not subjected representatives of the genus 
at his disposal to the same protocol used with i^gj aegypti , and, therefore, 
this seemed an appropriate starting point for the present investigation. 
Kartman (1953a) had shown, using Cx. quinquefasciatus t and this had been 
corroborated by Nayar and Sauerman (1975). using Cx. nigripalpus , 
that the characteristic poor host efficiency, defined as the number of 
target tissue (Halpighian tubule) developing forms divided by the total 
number microfilariae ingested, was a function of low number of micro- 
filariae reaching the tubules, that the "barrier," manifest in the midgut, 
lay somewhere prior to the target tissue, since there was ample evidence 
in these species that there were no further barriers of significance 
once the target tissue had been reached. This contrasts sharply with 
the pattern exhibited by the Ae. aegypti ;D. immitis vector-parasite 
relationship, where the major barrier, involving arrest of development, 
awaits the microfilariae in the target tissue (Kartman, 1953a; Nayar 
and Sauerman, 1975). The possibility that Kartman's hypothesis might 
apply to Culex , where the prolonged midgut experience, resulting from 
being trapped in the clot, could either have sapped the strength of the 
microfilariae or subjected the microfilariae to contact with a substance 
which was with little effect when exposure was brief, was therefore 



77 

sizable. In addition, Cx. quinquefasciatu3 and Cx. nigripalpus vector- 
parasite relationships with D. immiti3 resemble one another with respect 
to rapid bloodmeal coagulation, hemolysis of erythrocytes and crystal 
formation (Nayar and Sauerman, 1975). Therefore, the hypothesis was 
tested using the basic additiomdeletion strategy initiated by Kartman 
(1953a) and expanded in this case, to uncover the mechanisms responsible 
for the low susceptibility level. 

In contrast with Kartman' s ( 1955a) approach, no attempt here was 
made to measure the actual speed of migration of microfilariae through 
the midgut to the Malpighian tubule3, because it had not yet been shown 
to induce a significant dislocation in the vector-parasite relationship, 
as measured by an increase in host efficiency or some other susceptibility 
parameter. Kartman, for example, had discontinued his anticoagulant 
experiments after the 24 measurement and therefore did not determine 
if the increased rate was associated with an increase in developing 
forms, although his own data indicated that this would be the appropriate 
parameter to measure, as a huge portion of microfilariae that attain 
the target tissue do not develop further, a3 has since been noted by 
Nayar and Sauerman (1975), who discussed the tubule barrier, and, using 
Brugia spp., by Beckett and MacDonald (1971 ) and Schacher and Khalil 
(1968), where arrest occurs in the thoracic musculature. However, for 
Cx. nigripalpus , the "host efficiency" parameter of Kartman ( 1955a) is 
ideal, since, as he puts it (1955b), "It is of interest to note that 
although Cx. pipiens was an unfavorable ho3t for survivorship of worms, 
if they did survive then it was a favorable host for their growth" p. 1068. 
Therefore, the number of prelarvae in the tubules relative to the number 
of ingested microfilariae (host efficiency) is a sufficient parameter to 



78 

detect dislocations from the normal vector-parasite relationship 
induced by the addition: deletion strategy. An. qu adr imacul at u a controls 
were fed simultaneously to insure that the absence of response was not 
due to loss of microfilarial vitality from the treatment, and the 
bloodfed + 24" assay time avoids having to contend with the additional 
variable of mosquito mortality associated with this species (Weiner and 
Bradley, 1970; Nayar and Sauerman, 1975? Hamilton, 1975)* 

One extremely important aspect of most filarial: arthropod relation- 
ships in terms of delineation of mechanisms is the individual variation 
in susceptibility level; although the existence of just such variation 
is discernable from the results of Kartman (1953a) and Nayar and 
Sauerman (1975) » while it has been alluded to in much earlier work 
(Hu, 1931; Yen, 1938). One is usually confronted with an array of host 
efficiencies, developing form ratios and infective potentials, with the 
frequency of any particular level of 3uaceptibility being characteristic 
for the species as a whole. For example, if even one individual of 
thousands dissected is positive for infective 3tages, the species cannot 
be categorized as totally refractory, and, on the other hand, if 5% 
of the individuals of a species are refractory to infection, the species 
cannot be labelled totally susceptible. 

It is apparent from the work of Kartman (1953a) and Nayar and 
Sauerman (1975) that the usefulness of the number of ingested micro- 
filariae as an estimator of future events, such as host efficiency, 
developing form ratios and the like, is variable depending on the species. 
While in a highly susceptible species such as An. quadrimaculatus , the 
number of microfilariae ingested is tightly correlated with the number 
of prelarvae (microfilariae in the Kalpighian tubules) and each successive 



79 

stage (with a decrement constant factor applied) found, this is plainly 
not so with either Qui ex spp. or Ae. ae^ypti . For example, 6O/0 of Cx. 
guinquefasciatus ingesting 376 - 42 microfilariae of D. immitis had only 
2-6 prelarvae per female, but some had none and others had more (Nayar 
and Sauerman, 1975); thus, the ingestion of more than 100 microfilariae 
usually yields few if any prelarvae, (thereby setting the pattern of 
low susceptibility by which both species are characterized) but on 
occasion can yield many prelarvae, far outside of the "general" pattern. 

Experiments and results 

To confirm that the number of microfilariae ingested by Cx. 
niflripalpus is indeed a poor estimator of prelarval load or "host 
efficiency," groups of mosquitoes were fed on the infectious dog; after 
24 » the midgut and tubules were removed, separated and counts made of 
midgut microfilariae and tubule prelarvae (Table H-l). The shallow slope 
and weak correlation coefficient indicate that there is little relation- 
ship between the number ingested and the number of prelarvae, which is 
intuitively obvious given that most values of Ingested microfilariae 
are associated with low to zero values of prelarvae. Similarly, the 
occasional highly susceptible individuals are not the result of a 
particularly high or low number of microfilariae ingested. Since any 
proposed mechanism mu3t explain the existence of various individual 
host efficiencies and how they are generated, it is clear that the 
mechanism is not merely the ingestion of large numbers of microfilariae 
producing an array of prelarval loads. 

Before initiating experiments using the addition: deletion strategy, 
the basic frequency distribution of individual susceptibility generated 



80 



by normal (untreated) Cx. nigri palpus feeding on the infectious dog and 
dissected no less than 24 later for prelarvae in the tubules, was 
determined (Table 11-2^ Less than 5;£ are "susceptible" by An . 
quadrimacul atu3 standards, and a tenth covers the percentage of individu- 
als carrying more than 10 prelarvae. The predominant group, as expected 
based on previous work with this mosquito (Hayar and Sauerman, 1975), 
accounting for 90% of this sample, is comprised of individuals harboring 
10 or less microfilariae (Table n-2) and it is this group upon which the 
general susceptibility level of the species is based. 

To test the hypothesis, first advanced by Kartman (1953a), that 
hindrance of movement of microfilariae through the midgut of the mosquito, 
caused by absence of salivary gland anticoagulant, might account for the 
observed low susceptibility level, his strategy, that is, addition of 
the variable factor to simulate the susceptible mosquito, was followed. 
Three anticoagulants — citrate, heparin, and HDTA ( ethyl enediamine 
tetraacetio acid) — were tested, by feeding 7-14 day old Cx. nlgripalpus 
via the membrane feeder. The distributions generated by this "addition" 
protocol did not differ significantly from that seen in the "natural" 
group (Table E-2)and, if the purpose of the addition was to simulate the 
An . quadrimacul atus vector-parasite relationship, it can be safely said 
that this was not achieved. Thus, either movement hindrance and entrap- 
ment were not factors playing a role in determining the characteristic 
susceptibility level, or the simulation of the anticoagulated An . 
quadrimaculatus bloodmeal had not occurred using the protocol. The 
blood in the membrane feeder was fluid throughout the feeding interval 
(2-3 , with a majority usually having fed during the first half hour), 
and to determine if the same state was maintained after entering the 



81 

mosquito, midguts were dissected from mosquitoes within 15 minutes 
after imbibition of blood containing each anticoagulant. Regardless 
of the anticoagulant, approximately 95\ ; of all meal3 were clotted to 
various degrees, indicating that the anticoagulant had failed to 
function a3 expected (Table II-3). Considering the difference in modes 
of action —both calcium chelation and binding (2DTA and citrate) 
and thrombin formation inhibition (heparin) (Simmons, 1968), this was 
quite provocative, and strongly implied the presence and activity of a 
coagulant which induced clotting even in anticoagulant-treated blood — 
the presence of coagulants in mosquitoes was reported by Buck (1937) • 
A second implication is that the salivary gland anticoagulant activity- 
level cannot be assayed in directly bloodfed Cx. nigripalpus , although 
the absence of such activity might easily be inferred from the compara- 
tive clotting times of /In. quadrlmaculatuo and Gx . nigripalpus (Nayar 
and Sauerman, 1975) f e d directly on hosts, and, on a generic basis, 
from the absence of activity having been reported in Gx. salinarius , 
Cx. quinquefasciatua and Cx. restuans (Metcalf, 1545 )» a "d Gx. pipiens 
(Yorke and Macfie, 1924), while low level activity was observed in 
Gx . pipiens (Buck, 1937. Hudson, 1964) anri Cx, tarsal is (Hudson, 
1964). Since no direct information on either salivary gland anticoagulant 
activity or midgut coagulants activity existed for Cx. nigripalpus , 
determinations were made for both entities. The clotting time of blood 
was clearly increased over controls (Table II-4), although thi3 effect 
would not be measurable in vivo , since clotting of bloodmeals in the 
midgut is nearly instantaneous (Table II-?). 

The discovery of salivary gland anticoagulant increased sub- 
stantially the likelihood that the rapid coagulation of ingested dog 
blood and ensuing hindrance of movement of D. immitis microfilariae 



82 

was a function of the presence of a coagulant rather than absence of 
anticoagulant, and a characterization of this component was therefore 
necessitated. A stomach secretion from Culiseta (= Theobaldia ) annul ata 
caused rapid coagulation of blood, even when the blood was treated with 
Anopheles salivary gland secretions ( Suck, 1937); the secretion was 
not present in the anterior midgut or in male midguts. A coagulin was 
present in Cx. pipiens , but in much smaller concentration. 

The initial assay tested the extract from 10 Cx. nigripalpus 
female midguts mixed with 50 p:l dog blood treated with 100 units sodium 
heparin per ml, 5 times the amount needed to maintain the blood in the 
liquid state indefinitely. In all trials, the blood coagulated within 
1 minute, with first threads detected within 30 seconds, almost 
instantaneously (Table II-5). 'alien the anticoagulant level was raised 
to 1000 units heparin/ml, again, rapid coagulation of the sample was 
observed. No effects on clotting times were observed using either 
An . quadrimaculatus or Ae. aegypti extract, even when 10 midguts were 
tested on blood treated with 10 units heparin/ml dog blood, which is 
right on the verge of clotting spontaneously. The coagulant effect 
did not wane until 0.5-1.0 midguts wero mixed with dog blood treated 
with 1000 unit 3 of heparin/ml (Table II-5). 

Discussion 

As Buck's (1937) results are not accompanied with quantitative 
data, a comparison of the strength of the coagulins in Cx. nigripalpus 
with those in Cu. annul ata or Cx. pipien3 i3 not possible, but the mode 
of action seems similar. The results here attest to the presence of a 
powerful coagulin, similar in strength to Culiseta and greater than that 
of Cx. pipiens , with a surprisingly versatile chemioal property, in that 



83 

it can override the anticoagulant properties of substances with 
different modes of action. The mode of action of this coagulant remains 
to he determined, and it cannot be stated that it induces or is involved 
with the normal "cascade" of coagulation. There were indications that 
citrate could prevent the effect when used in higher concentrations, 
but in vivo tests were frustrated by refusal of mosquitoes to feed on 
blood thusly treated. 

Thus, the addition of anticoagulants, in the levels used here, 
failed to satisfy the requirement of the addition: deletion strategy, 
namely that the equivalent of an An . quadrimaculatus bloodmeal, liquid 
in state and offering little hindrance to trophic movement by micro- 
filariae, be produced in Cx. nigripalpus ; the meal did not remain 
liquid, a clot being formed as in "normal" mosquitoes, and movement 
hindrance from coagulation was not prevented. The basic vector— parasite 
relationship, as measured by the prelarvae per female frequency distri- 
bution, remained essentially unchanged. Although the proper biochemical 
characterization of the coagulant is beyond the aims and 3cope of this 
investigation, 2 lines of evidence suggest that it has a protein com- 
ponent: de Buck's (1937) characterization of the Culiseta coagulant-** 
unaffected by prolonged drying, heating to 99 0, heating of the dissolved 
secretion at 40 G for 30 minutes; dissolved secretion deactivated by 
50 C for 15 minutes - suggests this, and, in the present work, the use 
of glass-bead defibrinating of blood, which satisfied the requirement 
that a low-hindrance bloodmeal be induced in Gx. ni/yripalpus , supports 
a contention that fibrinogen is the protein involved. Later, tests on 
serum collected from blood incubated in thrombinized vials for 1 at 
37°C (Sigma Chemical Co. Bull. No. 850, 1977) proved negative for clotting. 



84 

Initially, the use of the glass-bead dofibrinating process had 
been rejected in favor of the addition of exogenous anticoagulant for 
2 reasons: 1.) the purpose of the protocol was to simulate, as nearly 
as possible, the An. quadrimaculatus midgut environment, and, while 
there may be little chemical affinity between a polysaccharide such as 
heparin and proteinaceous salivary gland anticoagulants, the mechanism 
whereby the coagulation cascade is interfered with is essentially 
similar and the net result, the absence of fibrin formation, is the 
same, while glass-bead defibrinating does not interfere with the 
cascade, since it merely prevents clotting by disallowing continuous 
fibrin strand formation. 2.) The very procedure of glass-bead de- 
fibrinating, where the blood is "churned" with beads for JO minutes, 
seemed more likely to traumatize microfilariae. However, with the 
failure of the anticoagulants to function as required, the testing and 
use of glass-bead procedure was mandated. Saponized droplets of blood 
samples taken following churning were examined for moribund or abnormal 
microfilariae but none were seen, and Giemsa-stained microfilariae on 
thin smears were not visibly wounded, indicating that the preparation 
was suitable for use in the membrane feeder. 

In the interests of volumetric equivalency, the dilution factor of 
0.1 ml of 0.9% Had, used in all anticoagulant experiments to dissolve 
the anticoagulant prior to mixing with blood, was also added to the glass- 
bead defibrinated blood. While approximately the same number of micro- 
filariae were ingested per female as in the anticoagulant-treated 
groups (Table II-2), more than twice as many mosquitoes had An. 
quadrimaculatu3- level prelarval loads (Table II-2), and the portion of 
totally refractory mosquitoes decreased dramatically. Nearly 30% of the 



85 

total number of mosquitoes fed on glass bead defibrinated blood had 
prelarval loads in the "moderate" susceptibility level category (Table 
II-2). A similar but smaller-sized shift of the distribution from the 
refractory and low susceptible categories to the moderate-but not 
upper— level categories had been observed in anticoagulant-treated 
groups but there was a greater than %'o chance that such a group could 
have come from the same population as the "natural," directly fed 
group, while the glas3 bead defibrinated group differed significantly 
(p<5/°) fr °m the directly-fed group (Table II-2)* 

Conclusions 

The above experiment gives "qualified" support to the hypothesis 
first advanced by Kartman (1953&) and supported by later work (Nayar 
and Sauerman, 1975), that the hindrance of microfilarial movement is 
a factor in determining the susceptibility level of mosquitoes to 
filarial infections. The mechanism for this hindrance in Cx. nl/^ripalpus 
is the rapid clotting of the ingested bloodmeal, due to the action of 
a powerful midgut coagulin, whose net effect was circumvented using a 
physical technique. However, the mechanism accounts for only a portion 
of the poor host efficiency characteristic of Cx. niflripalpus . The 
mean preiarvae per mosquito, while differing significantly from the 
mean of the "natural" group, implying that the treatment produced a 
real effect, was nevertheless still only in the moderate range. Thus, 
while the qualities of the An. quadrimaculatus bloodmeal had been "added" 
to the Cx. ni/rripalpus tD. immitis vector-parasite relationship, the 
susceptibility level of this sample' 3 population, while greater than 
that of the "natural" group, was far lower than that of the "susceptible" 
model, An. quadrimaculatus , implying that either other aspects of the 



B6 

hindrance faotor were not being addressed by the design or that other 
mechanisms were operating in addition to those associated with movement 
hindrance. 



'•7 



The Relationship of Hemolysis and Crystal Formation to Susceptibility 
of Cx. nigripalpus to Infection by D. immitis 



Introduction 

Another distinguishing feature of the Cx. nigripalpus iD. immitis 
vector-parasite relationship is the hemolysis of ingested red blood 
cells and subsequent production of needle-shaped oxyhemaglobin crystals 
in the bloodmeal (Nayar and Sauerman, 1975). Hemolysis of erythrocytes 
from Foley ella infected frogs occured in the midguts of Cx. territans 
within 20 min. after ingestion (Benach and Crans, 1975)* leading the 
authors to state "lysis of red blood cells and microfilarial death 
shortly after ingestion suggests barriers related to salivary gland or 
midgut secretion or to the mechanical action of the bucco-pharyngeal 
armature." With Cx. pipien3 t three types of secretions follow blood 
feeding (Arnal, 1950), and it was concluded that lysis of red cells was 
the result of metabolites or enzymes from bacteria that penetrated the 
meal. It is unlikely that hemolysis i3 the result of midgut digestive- 
proteolytic activity, which peaks at 24 after feeding (Gooding, 1972), 
and protease activity graphs (Briegel and Lea, 1975) show little or no 
activity during the first hour post-blood feedings, long after hemolysis 
has occurred. This does not rule out the possibility of a hemolytic 
agent potent at extremely small concentrations being present. However,, 
no hemolysins have ever been detected in mosquito salivary gland prepara- 
tions, even when specifically assayed for it (Gooding, 1972). 

In preliminary experiments, crystals were observed even in the 
bloodmeals of Cx. nigripalpus membrane-fed on glass bead-defibrinated 
dog blood (GBDF) and anticoagulant-treated dog blood, and the possibility 



88 

that they could, through their presence or absence, mediate the 
susceptibility level, through impedence of micro filarial migration or 
inflicting of wounds, made mandatory a more thorough examination and 
characterization of the phenomenon. The released hemaglobin it3elf 
radically changes the chemical environment in which the ingested 
microfilaria finds itself. 

Results and Discussion 

To determine whether the crystals were involved in mediating the 
vector-parasite relationships, crystals were deleted by membrane- 
feeding Cx. ni/yripalpus females, along with An . quadrimaculatus vitality 
controls, on anticoagulant-treated chicken blood, to which microfilariae, 
separated from an equal volume of infectious dog blood, had been added. 
Since the addition of anticoagulant to dog blood had not demonstrably 
affected the vector-parasite relationship in general (Table II-2), or 
coagulation time in particular (Table II-3), and, since the coagulation 
time of chicken blood in Cx. nigripalpus is even faster than dog blood 
(Nayar and Sauerman, 1975) » any increase in the portion of 3usceptibles 
or even moderate susceptible3 could bp attributed to either the absence 
of crystals, as chicken hemaglobin does not crystallize, at least not 
so as to produce observable entities under light microscopy within the 
time frame considered relevant here, or to the other variables attributable 
to differences in blood composition. 

Although there is an indication of some microfilarial vitality los3 
a3 seen in the An, quadrimaculatu3 control group (Table II-2) (most likely 
due to the use of the distilled water microfilarial separation method, 
later improved upon by using saponin), there was no indication whatever 



39 

that the absence of crystals improved the susceptibility profile 
(Table II-2); the same large portion of totally refractory and low 
susceptible mosquitoes dominates the profile. While these results are 
not, by themselves, conclusive, because of the possibility of un- 
controlled variables from the microfilarial separation procedure or 
blood composition that could differentially affect the reaction in 
Cx. niftripalpus compared with An. quadrimaculatus , they do not support 
the lethal-crystal formation hypothesis. However, an interesting 
phenomenon was observed during the course of bloodneal examination for 
crystals; instead of the usual picture seen with dog blood, where one 
observes red cells, red fluid and crystals, 3U3penaion of the chicken 
bloodmeal in normal saline revealed the presence of large numbers of 
2 u diameter spheroids. That these were indeed chick red cell nuclei 
(the erythrocytes of bird blood are nucleated) was confirmed by ob- 
serving the hemolysis of 3X washed chicken erythrocytes under a coverslip, 
running distilled water (later saponin) under one side of the slip with 
a fine capillary. Apparently, the nuclei are not at all subject to 
hemolysis, by either distilled water or saponins, since 50 >il of washed 
cells in 15 ml distilled water, followed by centrifugation, produces 
a pellet of nuclei. This accounts for the observation that hemolysis of 
chicken blood does not produce the clear, transparent red solution seen 
with dog or human blood; instead, a cloudy, only slightly less opague 
suspension is seen and clear fluid is produced only by centrifugation. 
The significance of this phenomenon was that hemolysis had occurred and 
had left a marker other than released hemaglobin, that thi3 marker was a 
measurable entity without having to run a laborious procedure, and 
without the need to calibrate for bloodmeal size. 



90 

A baseline hemolysis rate profile was established by feeding 
Cx. nigripalpus on the chicken and dissecting meals into saline at 
intervals from to 8 . An array of hemolysis values was generated, 
including a few extensively hemolyzed bloodmeals, a few with nearly no 
hemolysis and a majority with 65 - 15-j in preparations dissected at 
30 min. post bloodfeed and later (Table II-6). Zero hour (0-15') 
hemolysis was less extensive, suggesting that hemolysis was progressive 
with time and therefore a function of duration of exposure to the midgut 
environment. However, Giemsa-stained thin smears of zero hour blood- 
meals revealed the presence of many cells with fissures and reticular 
vacuolization, not seen in later preparations, and therefore indicating 
a probable pre-hemolytic condition. To test thi3, readings from hour 
preparations were left at room temperature and reread at +1 hour (Table 
II-6). A distinct increase in average hemolysis rate was observed. 
In saline, cells in the pre-hemolytic condition acquire a rounded versus 
normally oval shape and are considerably paler, probably due to loss of 
heraaglobin. Only very low levels of hemolysis were detected in Anopheles 
and Aedes controls. Histologically, the three species present distinct 
pictures (Figare II-e). The clear areas in Aedes and Anopheles blood- 
meals contrast sharply with the ubiquitous occurrence of a diffuse red 
staining material, probably fixed hemaglobin, in Culex. 

The basic pattern of hemolysis was verified by feeding Cx. nigripalpus 
on glass-bead defibrinated chicken blood (GBDP), thereby effectively 
circumventing coagulation itself and unmeasured variables introduced by 
the clot-squash procedure. This procedure (GTCDF) had already been 
determined to be fairly harmless to microfilariae (Table II-2), and the 



91 




Figure 8a 




Figure 8b 

Figure II-8. Serial sections of a) anterior midgut of Cx. 
nigripalpus and b) posterior midgut of Cx. nigripalpus , 
showing free nuclei from hemolyzed cells, and c) posterior 
midgut of Ae. aegypti , showing agglutination but no hemolysis; 
Hematoxylin-eosin; 14u thickness; x400x. 



92 




Figure 8c 



93 

same was true regarding chick red cells, since control values (Table 
II-6) were again indicative of minimal hemolysis. The basic profile 
was essentially the same with slightly less variability, but, clearly, 
coagulation phenomena have nothing to do with the hemolysis phenomenon, 
at least in the time frame considered here. 

That the interval from bloodf ceding to observable hemolysis in 
Cx. ni/rripalpus is so brief essentially rales out digestive enzymes as 
the causative agents of hemolysis, but doe3 not rule out the action of 
other chemicals, produced by the vector or its flora, encountered in 
the bloodmeal. However, the likely sources of such chemicals are 
limited by physiological considerations to the salivary gland lobes 
and midgut epithelia, tissues with known secretory properties. Although 
the fact that salivary gland substances with hemolytic properties have 
not been demonstrable even when specifically sought after (Gooding, 
1972) suggests that they most likely emanate from the midgut epithelium, 
because of the sizable variation in digestive processes involving 
salivary gland secretions from mosquito to mosquito fed on the same 
blood source and, within a species, variation in processing after feeding 
on different blood sources (Metcalf, 1945), extracts of salivary gland3 
were tested for hemolytic activity. Neither fresh cover3lip squashes of 
glands in the presence of chick RBC or concentrated extracts produced 
demonstrable hemolysis (Table II-7). 

Midgut extracts, prepared as for coagulant studies, did not induce 
hemolysis of chick red cells, even when incubated in strong concentra- 
tions for one hour (Table II-7)« t\irthermore, fresh centrifuged 
bloodmeal liquid was also without effect, a3 was injection of red cells 



94 

into serum-fed Cx. nigripalpus (Table II-7). Finally, in vitro 
inoculation of midguts, via the cardiac valve (Table II-7) and in vivo 
per ano enema with red cells in serum or saline, failed to produce 
significant (above baseline) hemolysis, with the exception of a few 
isolated cases possibly induced by exposure to hindgut or crop contents 
with hemolytic pH levels (Table II-7)« Crops contents of Gx. nigri palpus 
measured in this investigation were often as low ae pHJ, and hemolysis 
of red cells occurred when mixed with these (Table II-8). 

The net inference from these experiments is that while hemolysis 
might first be manifest and demonstrable in the midgut, it is actually 
induced at some point prior to the midgut, while salivary gland fluid 
might serve as potentiator, no hemolytic activity was demonstrable with 
either salivary gland extract or midgut extract, including midgut 
secretotogues from enema or cardiac valve procedures, both of which 
distend the midgut to a point where the secretotogue effect could be 
expected to occur. It is of course provocative that the absence of 
hemolysis occurs at a rate similar to the rate of prelarval susceptibility. 
Thus, while hemolysis-crystal formation and microfilarlal vitality might 
not be casually related, they are related temporily and in terms of 
rate; hemolysis is therefore, at least an indicator of an timicro filarial 
phenomena in this mosquito. 



95 

Evaluation of Alterations in Susceptibility Level of Cx. nigripalpus 
to D. immitis infection Induced by Additives in the Blood Weal 

Introduction 

The failure of exogenously administered anticoagulant to prevent 
the characteristic rapid clotting of the ingested bloodmeal in the 
midgut oi' Ox. nigripalpus females (/fable 11—3 j was not devoid of benefit, 
as it made possible the introduction of a variety of substances into 
the mosquito during bloodfeeding by membrane, which could not otherwise 
be so introduced without hazard to the definitive host. The very 
inability of anticoagulant to meaningfully dislocate the Cx. nigripalpus : 
^* inimitis vector-parasite relationship meant that the effect of other 
substances could be determined without having to contend with a multi- 
plicity of variables; the blood food, which remains liquid in the 
feeder due to the action of the anticoagulant, is processed by the 
mosquito just as if it were a "normal" meal, and, therefore, the 
observed effects are attributable to the presence of the "additive." 
The hemolysis oi* red cells and morbidity of filariae, seen in both the 
Cx . territans s Foleyella brevicauda (Benach and Crans, 197!? J and Ux. 
n igripalpus i D. immitis l^Nayar and Sauerman, 1975 ) vector-parasite 
relationships, suggested that chemical agents produced in the normal 
course of digestive processing might be responsible; thus, interference 
with or disruption of these normal processes by alteration of the blood 
composition offered a means of dislocating the susceptibility level in 
a way that would offer revealing insights into the responsible mechanism. 
It is well-established that most enzymatic reactions are optimal under 
very specific conditions, which, if altered, induce changes in rates of 
reaction in the system. That different species of mosquitoes process 



96 



blood from the same animal differently, and that within some species, 
blood from different sources is processed differently in terms of 
agglutination and coagulation, (Metcalf, 1945; Hudson, 1964 ), 3ugge3ts 
that the digestive process is variable and dependent on substrate, 
(Breigel and Lea, 1975)* The susceptibility of Aedes aegypti to 
infection by Plasmodium gallinaceum wan altered by administration of 
organic and inorganic acids bases and salts (Terzian, 1958 5 Terzian and 
Stabler, i960), plant extracts and drugs (Johnson and Akins, 1943); 
in some of these cases (Terzian and Stahler, 1964), while the substances 
were affecting the normal digestive processes in such a way that the 
host-parasite equilibrium was perturbated, the observed effects could 
not be linked with a specific inhibitory mechanism. Hovanitz (1947)t 
studying the same system, divided physiological factors that might 
mediate susceptibility levels into 2 categories, those of hereditary 
nature, such as species and strain specificity which can be selected 
for, and those of environmental nature, such as, individual variability 
within a strain, although distinguishing between the 2 comes down to 
whether or not the factor can be selected. The vector-parasite relation- 
ship in the same system was altered from "normal" by feeding mosquitoes 
whole body extracts made from susceptible and refractory mosquitoes 
(Weathersby et al . , 1971), and, along with studies on the development 
of Plasmodium gallinaceum in the hemocoels of refractory and susceptible 
mosquito species (Weathersby and MoCall, 1968), suggested that undetermined 
toxic factors were probably responsible for the resultant deterioration 
and death of the parasites, although such "antiblastic" toxicity effects 
were also observed in groups of susceptible mosquitoes, making specific 
interpretations of mechanisms difficult. It seems quite possible that 



97 

all of the foregoing effects were manifestations of undirected perturba- 
tions in the physical -chemistry of the mosquito midgut. It should be 
noted that in all of the experiments just described, the substances 
were introduced in the sugar meal and were therefore subjected to a 
different processing protocol than would be the case if they were in 
the blood food. When blood fractions were introduced during the blood- 
feeding process (Behin, 1968), certain proteins, such as albumin, were 
efficacious in producing shifts in susceptibility level in the same 
system, but, again, the mechanism whereby thi3 occurred could not be 
sharply delineated. Terzian and Stahler (i960) concluded that the 
results indicate that the binding of ion3 to specific sites accounts 
for the observation that substances active singly were not more active 
in combination, that concentration was more important than pH effects, 
since the same substance could increase or decrease susceptibility 
depending on its concentration. The same inferences were made regarding 
the action of antibiotics, vitamins and hormones (Terzian et al ., 1953; 
Terzian, 1958). 

In the present study, consideration had to be given to effects of 
any particular additive on microf ilarial vitality and therefore vitality 
controls, in the form of An. quadrimaculatus , were run simultaneously 
with experimental groups. Buffers had been shown by Terzian and Stahler 
(I960) to be capable of producing susceptibility shifts most likely 
attributable to disturbance of midgut physical -chemistry, and, since it 
was the purpose of this experiment to induce changes in the susceptibility 
level that could be associated with perturbations of the digestive 
process, buffers were also chosen as additives in this case. However, 
while Terzian and Stahler (i960) employed relatively large concentrations' 



98 

(100 mM), levels used in this study were based on concentrations found 
in various buffered saline3, such as described by Nijhout and Carrow, 
(1978), and are therefore in the 2-8 mil range. Complete groups could 
not be easily attained in many cases, due primarily to palatability 
problems. Both inorganic phosphates and organic, "zwitterionic" 
"Good" buffers were added, the latter because of absence of precipita- 
tion of polyvalent cations and low toxicity and known efficacy in ti33ue 
culture systems (Good et al ., 1966; Eagle, 1971). Both types of buffers 
are relatively uncomplex substances with molecular weights mostly below 
200, compared with other active substances such as hormones and vitamins 
(Terzian et al ., 1953), making interpretation of observed physiological 
changes or alterations in susceptibility level;:, less difficult. Buffers 
were added with 3 different anticoagulants — citrate, EDTA, and heparin. 

Results and Discussion 

Highly significant dislocations in the vector- parasite relation- 
ship between Cx. nigripalpus and D. immitis were produced by the addition 
of some of these buffers to the anticoagulant-treated blood food (Table 
II-9); the magnitude of this dislocation depended on the anticoagulant 
and the buffer. While the perturbation induced by citrate acting alone 
did not differ significantly from that of the other anticoagulants 
(Table II-9), in conjunction with certain buffers, especially "TRIS," 
potassium biphosphate, "CAPS," "ACES," "BICIHE" and sodium biphosphate, 
the number of mosquitoes harboring "highly susceptible" level infections 
increased by a factor of 10, and both 0.375 mg/ml and 0.75 mg/ml TRIS 
increased the number of "highly susceptibles" by 5X (Table II-9). 
While of smaller magnitude than in the citrate group, similar changes 



99 

in susceptibility level occurred in the EDTA and heparin groups. The 
addition of monobasic potassium phosphate induced statistically signifi- 
cant increases in susceptibility in all 5 anticoagulant groups, while 
TRI3, efficacious with citrate and EDTA, was without effect when used 
with heparin. Preconditioning mosquitoes by addition of the buffer, 
in the same strength, to the 10/j sucrose maintenance solution, confirmed 
the efficacy of potassium bi phosphate (monobasic) in generating a group 
of mosquitoes of significantly greater susceptibility than untreated 
controls (Table 11-10). However, an increase in the susceptibility 
level of the citrate-only group preconditioned with citrate in sucrose 
was provocative, while again the heparin-TRIS combination was ineffectual 
(Table 11-10). No groups fed directly on the infectious dog, regardless 
of preconditioning regimen, produced significant dislocations in the 
vector-parasite relationships. 

The variability in physic-chemical character of the efficacious 
agents, especially between monobasic potan3ium phosphate, the addition 
of which decreases the pH from normal, and TRIS, which raises it, 
supports the contention of Terzian and Stahler (i960) working with a 
variety of substances and their effect on susceptibility of Ae. aegypti 
to Plasmodium gallinaceum , that since these compounds represent wide 
extremes in the range of pH it appears reasonable to assume that these 
effects on host susceptibility are due essentially to 1he action of the 
particular ions rather than to the influence of pH in itself, and 

changes in susceptibility or resistence seem to depend upon the particular 
salt, and the concentration in which it i3 administered, rather than any 
specific changes in pH. In the present case, whether a specific aspect 
of the digestive process is affected, or the entire meal is processed 



100 

differently due to different information being detected by the insect 
nervous system, cannot be determined. The complex interplay between 
ingestion and processing of bloodmeals and the nervous system's coordina- 
tion of a variety of resultant events, e.g., oogenesis, is only now 
beginning to be understood. Sensilla located in various parts of the 
alimentary canal, especially the buccal cavity, probably respond to 
various qualitative and quantitative factors (temperature, osmotic 
pressure, salt and sugar levels) in the meal, coordinating processing. 
The objective of these experiments, to dislocate the vector-para3ite 
relationship, had been accomplished, although the mechanism to account 
for this dislocation remained obscure. 



101 



The Determination of Sources ofMlcrofilarial Vitality Loss 

The loss of vigor and death of microfilariae of D. immitis in the 
midgut is a salient feature of the Cul ex ; onchocercid microfilariae 
vector-parasite relationship. Hu ( 1931 ) » discussing variable suscepti- 
bility determinants in mosquitoes to D. immitis infection, found 
degenerate microfilariae, immobile and extended to their full length, 
in the midguts of Cx, pi pi ens 2 day 3 after feeding, and O'Connor and 
Beatty (1938) mention feeble microfilariae of D. immitis in Cul ex 
midguts, while Travis (1947) noted that nearly all microfilariae larvae 
that remained in the stomach of Cul ex on the second day were dead, 
whereas a high percentage was still alive in Aedes. Kartman ( 1953a), 
investigating the fate of D. immitis in the midgut of 6 species of mos- 
quitoes in 3 genera, found dead, intact microfilariae in the Cx . 
quinquefasciatus midgut by 8 hours after infection and 12 hours in 
Cx . pipiens , with a majority of microfilariae being killed by 24 hours 
after infection, and he hypothesized that a factor in the midgut or 
salivary glands of Cx. pipiens and Cx. quinquefasciatus , not present 
in Ae. aegypti and A. albopictus , is capable of rapidly killing the 
microfilariae prior to their digestion. At 1 after bloodfeeding, 
dead microfilariae of D. immitis were found in the midguts of Cx . 
salinarius (Seeley and Bickley, 1974) and Cx. nigripalpu3 (Nayar and 
Sauerman, 1975)* Similar patterns in vector-parasite relationships 
involving Culex and other filariae have been reported; most ingested 
Brugia pahangi remained in the midgut of Cx. quinquefasciatus , and only 
a fourth of these were alive but were always feeble (Bwert, 1965)» 
while less than 2/'o of Foley ella flexicauda were able to escape the midgut 



102 

in Cx. territans (Benach and Grans, 1975 )t and the majority of micro- 
filariae were dead in the midgut, leading the authors to state "this 
barrier may be related to either digestive secretions of salivary 
glands and midgut or to mechanical action of the bucco-pharyngeal 
apparatus, or both" p211. In pipiens , 79 "^ ingested Brugia pahangi 
died in a susceptible strain, while 99/j died in the refractory strain 
(Obiamiwe, 1977)* Damage to microfilariae of Dirofilaria repena found 
in midguts of Qui ex pipiens was attributed to the action of the bucco- 
pharyngeal armature, since damage did not occur in those species lacking 
the structure (Ooluzsi and Trabucchi, 1968), and others have essentially 
repeated and confirmed this with Ox. fatigans infected with Brugia and 
Wuchereria , (Bryan et al . , 1974; KcGreevy et al. , 1978). The purpose of 
the present investigation was to determine whether the observed I033 in 
microfilarial vitality was a function of the presence of antiblastic 
factors in the Cx. nigrlpalpus midgut, physical damage to the larvae, 
or both. 

The quantitative analysis of microfilariae vitality in the midgut 
of different species of mosquitoes wa3 first done by Kartman (1953a), 
who established the following categories: normal; alive but abnormal; 
dead and intact; and dead and disintegrating. KcGreevy et al . , (1978), 
in addition to assessing actual damage (wounds) to microfilariae of 
Wuchereria and Brugia, measured vitality dynamically using a motility 
index, whereby a microfilaria wa3 scored as amotile if it didn't move 
for 15 seconds. In thi3 study, vitality wa3 assessed using a hemacytometer- 
type chamber after the method of Biles (1973) and damage assessed from 
specimens fixed and stained after the method of Awogun (1978) • In 
addition, a brief analysis was made of damage location on the microfilaria, 



103 

and vitality I033 was measured as absence of motility or abnormal 
undulating movements, including 1 spastic, jerking movements and sharp, 
angular motions around a single point on the body, easily discernable 
from the smooth, rhythmic undulations of a normal filaria, 

D. immitis microfilaria from the midguts of Cx_. nigripalpus fed 
on glass-bead-defibrinated chicken blood to circumvent clotting, ex- 
hibited a iv,ige of vitality loss, from none to total, depending upon 
the mosquito from which it was taken (Table 11-11 ). Host mosquitoes 
harbored microfilaria! loads in which more than half were moribund, 
but only a small portion of mosquitoes harbored totally unaffected or 
totally moribund micro filarial loads. The precise value again varies 
with the individual and any single characterization of the species 
as a whole with regard to vitality loss must, therefore, be a generality, 
and this is the usual case for Qui ex spp. as seen in the literature. 
The morbidity patterns explain the poor host efficiency of the species 
as a whole as measured by the prelarvae per female assay (Table II-2). 

Since previous experiments in this investigation supported the 
hypothesis of Kartman (1953 a )» that hindrance of migration of micro- 
filariae to the target tissue was involved in determination of suscepti- 
bility level, the possible influence of salivary gland or midgut 
secretions or microfilarial vitality was therefore investigated. That 
vitality level wa3 a function of the occurrence of events prior to the 
midgut or early in the midgut was suggested in the results of per ano 
injections of Gx„ nigripalpus (Table II-ll), since there was essentially 
no loss of vitality relative to the bloodfed control. While this 
procedure attempts to "delete" anterior alimentary canal and salivary 



104 

gland factors, it does not rule them out of "potentiator" roles, 
acting in concert with another component. Nevertheless, the fact 
that microfilariae so introduced to Ox. ni/rripalpus are indeed 
"infecting" the mosquitoes, shown by the subsequent migration to and 
development in the Malpighian tubules (Table 11-11 ), is strong evidence 
that determining events are occurring early in the midgut or prior to 
the midgut, even if their manifestation is in the midgut itself. This 
is supported by experiments in which non-coagulable bloodmeal3 from 
just-fed mosquitoes were inoculated with D. iminitis microfilariae and 
in in vitro experiments where the liquid fraction from centrifuged 
bloodmeals was inoculated with microfilariae. Again, the absence of 
vitality loss was suggestive of factors exerting their action prior 
to or early in the midgut experience, ,/h.atever the factor was, its 
effect was not transient, and not a function of continued exposure to 
a factor, since rejuvenation experiments indicated that the morbidity 
was permanent (Table Il-ll). Absence of vitality loss in microfilariae 
inoculated into Ox. nigripalpus midguts via the cardiac sphincter in 
fresh in vitro preparations of alimentary canals, intact caudad from 
the proventri cuius, again supported the pre-midgut or potentiated 
midgut factor hypothesis. 

While "deletion" of the salivary gland variable by duct ablation 
or gland removal would perhaps have been the ideal procedure, duct 
ablation not only proved difficult with this npecios but al30 produced 
mosquitoes that were subsequently unable to feed. ".Deletion" of the 
midgut was not possible for in vivo experimentation, and, therefore, 
extraots of both organs were tested for the presence of antiblastic 
factors, which, in terms of addition:deletion strategy, deletes everything 



105 

but the organ. Both salivary gland and midgut extracts were prepared 
exactly as had been done for experiments which demonstrated the 
presence of active factors, namely, salivary anticoagulant and midgut 
coagulants, and were thus of proven biological activity; this does not 
rule out factors neutralized by the in vitro process itself, that is, 
processes that can occur only in the vital framework. Nevertheless, 
these preparations were without effect regarding microfilarial vitality 
compared with the baseline bloodfed control (Table 11-11 ). 

Fixed and stained smears of microfilariae from the midguts of 
bloodfed Ae. aegypti , An . quadrimaculatus and Cx. nigripalpus revealed 
that significant observable wounding occurred only in Cx. nigripalpus , 
although an occasional wounded microfilaria was found in Aedes aegypti 
(Table 11-12). The rate of wounding was not constant in all Cx. 
nigripalpus but wa3 again variable and therefore distributed (Table 
11-12). In mo3t (80>) mosquitoes, a largo portion of the microfilarial 
load was injured, but occasional non-wounded specimens were always found, 
and a small portion of mosquitoes harbored only normal microfilariae. 
As in McGreevy et al ., (1978) the vitality assay results were in 
general agreement with the wound assay results. Slightly lower 
vitality loss rates compared with wound rates could have occurred due 
to the failure to count affected microfilariae which if viewed a few 
hours later would have more strongly exhibited the characteristic 
symptoms, while the wound assay could miss truly mortally wounded 
microfilariae that simply do not show observable wounds, as measured 
here. The location of wounds on any particular part of the micro- 
filarial body was essentially equally distributed, no one section 
receiving a disproportionate number of wounds (Figure 11-10). What is 



106 



Percent 



II 



III 



:/ 



, 



VJI 




Figure 11-10. Distribution of wounds in microfilariae from Cx. 
nigripalpus . 



107 

of particular interest is the observation that, while most micro- 
filariae have only 1 wound, some have 2, and the implications of this 
will be discussed later. 

If the salivary glands and midgut are without a role in determining 
microfilaria! vitality, as the results indicate, and if most of the 
vitality loss can be attributed to wounding, the inference can be made 
that the vitality loss is due to injury from the bucco-pharyngeal 
armature of Coluzzi and Trabucchi (1968) and the following experimental 
strategy was employed to investigate this possibility. 



108 
Mode of Action of the Buccopharyngeal Armat ure 

•While the general host efficiency of Cx. nigripalpus for D. immitia 
is very low (Nayar and Sauerman, 1975), with a high percentage of 
individuals reducing the target tissue load (infection level) either 
totally (10C96) or by 95-99? / o, nevertheless, a majority of mosquitoes 
harbor at least one prelarva, indicating that the mechanism is not 
absolute, and an infrequent mosquito (1/10 - 1/20) carries an An. 
cuadrimaculatus- level infection (50 pi?), indicating that the mechanism 
can fail altogether from time to time (Table II-2). The use of 
certain buffers significantly increased the portion of such individuals, 
(Table II-9), although no consistent attribute of the buffers used 
could be associated with the effect, and the results of the wound assay 
indicated that while large numbers of HF were wounded in a majority of 
cases, it was nevertheless variable, with occasional individuals 
harboring no wounded MI* (Table 11-12), and, likewise, in the vitality 
assay, an occasional mosquito exhibiting no defense at this point 
(Table 11-11 ). Since the oxyhemaglobin crystals had been eliminated 
as a mechanism responsible for the wounds (Table 11-2), and other 
experiments such as enema suggested that the event took place prior to 
the midgut, the probability increased that the bucco -pharyngeal 
armature (Sinton and Govell, 1927) equivalent to the dorsal pharyngeal 
valve of Uchida (1979) and cibarial armature of McGreevy e t al., (1978), 
was responsible for the event. The classic study by Goluzzi and Trabucchi 
(1963), using Dirof ilaria repens, established that this armature was the 
likely source of damaged microfilariae, since the event occurred only 
in those mosquitoes which possessed it, including members of the genus 
Cuiex arid subgenera Cellia and Nyjssorhynchus in genus Ano pheles . The 



1C9 



percentage of damaged microfilariae appeared generally very high and 
accordingly the mosquitoes were found not infected or infected with 
very few larvae (1 or 2 instead of the 30-40 expected), and they 

concluded that the microfilariae are instantaneously damaged on their 
way through the buccol apparatus of the mosquitoes, and that the 
damage was "mechanical," cauued by the bucco-pharyngeal armature 
acting like a "toothed jaw." Bryan et al. f (1974) duplicated the 
results of Coluzzi and Trabucchi (1968) uning Bru/?ia pahanpri . a 
sheathed microfilaria, and IlcCreevy et al. t 1978, U3ing Voichereria 
bancroftl and Brugia pahan^i , again implicated the bucco-pharyngeal 
armature (their "cibarial armature") as the cause of wounds. However, 
they compare their results using Cul ex pi pi ens quinquefasciatus , with 
its weak cibarial armature composed of small delicate teeth, whose 
cibarial armature killed only 6",i of microfilariae, roughly equal to 
that in Ae. aefypti , stating that it is questionable if the cibarial 
armature of Cul ex is lethal at all, with the results of Coluzzi and 
Trabucchi (1968) who reported very high percentages, lOO/o in some 
cases, of damaged microfilariae, v/ith a reduction microfilarial load 
rate from 30-40 to 1. 

The possibility that the armature of Coluzzi and Trabucchi (1968) 
was also the straining ("sieving") mechanism of de /ioiasezon (see Day) 
(1930) was suggested in the results of Day (I954)i who investigated the 
mechani&m of food distribution in mosquitoes and determined that a 
"switching mechanism" existed which had its basis in the sensory system 
located in the cibarium (buccol cavity); these receptors, which could 
detect sugar, blood and plasma via chemical receptors and red cells via 
mechanical receptors, regulated the sphincters of the diverticulae and 



110 

midgut. The straining mechanism of de Boisaezon (1930) accounted 
for the presence of clear liquid in the crop and cells in the midgut 
after mosquitoes were fed on a suspension of sugar and cells} other 
experiments by Day (1954) and Hosoi (1954) demonstrated that the 
"switching mechanism" could be "fooled," even in Aedes aegypti , where 
it seemed strongest (Trembley, 1952), by feeding red cells suspended 
in sugar solution, resulting in their being routed to the crop. As 
Day (1954) states, ne occurrence of erythrocytes in the diverticulae 
when blood: sugar mixtures in other concentrations are fed demonstrates 
that the straining apparatus is not normally able to control the 
switching mechanism. Actually, the reverse of this situation more 
aptly describes the situation; that is, the switching mechanism, 
(receptors frontal ganglion sphincters) cannot always control the 
straining mechanism. 

If the straining mechanism were merely a static version of the 
"toothed jaw" or "ohewing action" of the operational armature, which, 
as suggested by Coluzzi and Trabucchi (1968), was connected with the 
rhythmic action of the pumps, the possibility existed that the normal 
operation of the armature, along with the sphincters, was capable of 
being deceived, altering the normal sequence of events wherein micro- 
filariae are wounded. If the straining mechanism was merely a "normally 
closed" armature during sugar feeding, opening only occasionally as 
particulate matter piled up against it, then the wounding percentage 
noted by Coluzzi and Trabucchi (1968) should be altered. This hypothesis, 
that the switching mechanism also controls the armature, and that the 
armature in Cx. nigripalpus is engaged only during the bloodfeeding, 
was tested by feeding mosquitoes on mixtures of microfilariae and 



111 

chicken red cells suspended in sucrose solution, (Kartman, 1953o, 
had fed An. quadrlmaculatus on MF suspended in 5% dextrose and found 
live MF in the crop) after a test of sugar solution:microfilariae 
compatibility revealed that 10% sucrose was a satisfactory medium 
(Table 11-13). Here, mosquitoes were fed u3ing the apparatus described 
in Figure II-7> after other techniques, such as "hanging drops" failed 
to insure ingestion of the mixture. Each tube was charged with the 
mixture, usually 50 ul, to a point where the downward pressure produced 
a flat surface from which the mosquitoes could feed. All mosquitoes 
were starved for at least 24 prior to being offered the mixture, and, 
after a few minutes in the cup ? most fed readily. 

That both Aedes ae/ypti and Culex nigrlpalpus males ingested red 
cells and microfilariae suspended in the sugar solution attests to the 
success of the system (Table 11-14). As expected, based on the results 
of Day (1954) » mixtures of chicken plasma, red cells, and D. immitia 
microfilariae, reconstituted to the original hematocrit, and 10% 
sucrose (yield 5% sucrose) were found in the gut, and wounded micro- 
filariae were found in all specimens (Table 11-12) that had ingested 
microfilariae, indicating that the sensory system was in a bloodmeal 
processing mode with in the "toothed jaw" operational. When chick 
red cells and microfilariae were suspended in 10% sucrose after 
washing in 10% sucrose, the meals went predominantly to the crop, in 
terms of volume, and often masses of microfilariae and red cells could 
be found in the crops; often the midgut also had a small volume ( 1 nl) 
of mixture in the midgut, and REG were mostly normal but with some 
evidence of hemolysis. Unexpectedly, if the hypothesis regarding 
inactivation of the armature during sugar feeding was correct, several 



112 

mosquitoes (36$) had morbid MF and hemolyzed cells producing a fluffy 
orange substance (Table 11-14). However, the appearance of the morbid 
and dead microfilariae was unusual in that they were either straight 
or in undulating posture, not the "kinked" position characteristic of 
wounded microfilariae, and there was no 3ign of abrasions. Samples 
of such larvae were stained as for wound assays, and examination 
revealed no wounds (Table 11-12), indicating that the armature was 
indeed inoperative during sugar feeding. Whether the microfilariae and 
chick R3C found in midguts was the result of the "weak" switching 
mechanism seen in other Culex (Trembley, 1952) or from movement of 
crop contents to the midgut soon after feeding, was not determined. 
The fact that the microfilariae were mostly normal (Table 11-15) 
favors the "weak switching mechanism" explanation. The pathology did 
not indicate mechanical injury but rather a chemical substance was the 
likely source of morbidity. Furthermore, when crops were dissected 
at 8 h post feed, the portion of mosquitoes with morbid microfilariae 
increased, suggesting microbial growth curve effects (Table 11^-15). 
Visibly liquid filled crops from 2-week old colony Cx. nlgripalpus , 
Ae . aegypti and An. quadrlmaculatus were removed to pH paper, on which 
they were ruptured under the dissectory scope. Nearly all crops had a 
pH less than 4.0 (Table II-8), and microfilariae were dead or moribund 
within 10 minutes after being placed in such liquid and could not be 
revived by flooding with Hank's saline} red cells hemolyzed within 30 
minutes although a 2 minute exposure insured eventual hemolysis. Crop 
contents from freshly emerged Cx. nigrlpalpus, imbibing their first 
sugar meal, were uniformly found to be approximately pH 6. 5. When fed 
on the mixture, such mosquitoes were not found harboring morbid micro- 
filariae (Table 11-15) or hemolyzed RBG (Table II -8). 



113 

In vitro Methylene blue staining of crop contents and Giemsa 
staining of fixed thick smears from older individuals revealed the 
presence of characteristic, threadlike hyphae and clusters of small 
spherical particles which stained measurements intensely; no attempt 
was made to culture these flora, but when 7-9 1-day old Cx. nigcipalpus 
were maintained on methyl-para-hydroxyamino-benzoate saturated in lCpd 
sucrose, and otherwise treated as were non-preconditioned controls, 
neither hemolysis nor morbidity (Table 11-15) occurred. Furthermore, 
mosquitoes 2-3 days post-emergence taking their first meal did not have 
moribund microfilariae or hemolyzed red cells (Table II-8). It is 
reasonable to conclude that the low pH of crops in older mosquitoes 
was responsible for the morbidity; that this was observed at all was 
coincidental, since the age group of Ox. n i^ipalp us used was selected 
as representative of that used in general for the other experiments, and 
this happened to be a 1-3 week interval. If only freshly emerged, first 
time feeding mosquitoes had been used, it is unlikely that the effect 
would have been noted. That the effect was less pronounced in the 
+2 dissected group suggests that the starvation period, intended to 
increase the avidity of the mosquitoes for the mixture, also reduced 
the level of microbe metabolic products responsible for lowering the 
pH, by removing the liquid growth medium; once the liquid is reintro- 
duced growth would start again, at a variable rate among mosquitoes. 
While not of relevance to the hypothesis regarding the operation of the 
armature, prelarvae were found developing in the Malpighian tubules 
of both sexes of Cx. nigripalpu3 . as well as Ae. aegypti and An. 
guadr i macul atus females (Table 1 1-9). 

While the armature has been characterized in Culex quinquefasciatus 
as "weak," composed of small delicate teeth, which killed less than 



114 

10#> of ingested Wuchererla bancrofti microfilariae (McGreevy et al ., 
1978) and these results are supported using Brugia malayi (Buse and 
Kuhlon, 1979), this same species killed or damaged most microfilariae 
of Dlrofllaria rep ens (Coluzzi and Trabucchi, 1968), and the 
differential response could not be attributed to miorofilarial length 
or presence of a sheath (McGreevy et al. t 1978). However, the 
armature of Culex pipiens pal lens is an impressive cluster of spines 
(Uchida, 1979) and appears well-suited to fulfilling a role as a 
lethal instrument. The armature in Culex nigripalpus likewise appears 
highly capable of inflicting wounds (Figure II-9) Thus, there appears 
to be considerable variation within the genus with respect to the size 
and shape of the armature, and, aside from its presence, few other 
generalizations seem applicable. 

The hypothesis that not only the sphincters but also the armature 
i3 receptor-controlled offers an attractive explanation for the 
results of the buffer experiments. When introduced with the bloodmeal, 
a variety of buffers at specific concentrations increased the number 
of mosquitoes with An . quadrimaculatua- level parasite loads with no 
vitality loss (Table II-9), indicating that the armature was more 
frequently inoperative in these groups. However, these meals went 
directly to the midgut, with never more than traces of blood in the 
crops; thus, the sphincters must have been set at the bloodfeeding mode, 
while the pharyngeal valve, the dorsal component of which is the 
armature, either remained open continuously, or opened and closed 
intermittently, as cells and filariae piled up against it, after the 
fashion of the straining mechanism (Boissezon, 1930). If the 
armature's operation is a part of the rhythmic oscillations of the 



115 



****** 





Figure II-9. Sections through buccal cavity showing dorsal 
pharyngeal valve ( bucco-pharyngeal armature); 14u thickness; 
heraatoxylin-eosin stain; x400. 



116 

pumping system, as suggested by Coluzzi and Trabucchi (I968), the 
absence of operation of the armature implies an absence of the operation 
of the cibarial pump altogether. The structure of the cibarium com- 
pared with that of the pharyngeal pump would indicate that the loss 
of function of the cibarial pump would not be of great importance. 
The alternative, equally conjectural in nature, is that the armature 
operates independently of the pumps, has its own sensory system, and 
is engaged, opened or closed independently. The buffers in such a 
scheme would either stimulate the armature chemoreceptors to a non- 
function mode or mask a component in the blood that makes the armature 
operational. Detailed histological studies combined with physiological 
techniques to manipulate the receptor system would likely resolve this 
problem. 

That the armature can kill as many microfilariae as it does, and 
tMs rises to 9$% in An* gambiae (McGreevy et al. t 1979) » suggests 
that the structure must be oscillating at an enormous rate, since the 
lumen at the point of the armature is very narrow and the flow rate 
must reach a relatively high velocity, just on general considerations 
of physics. If 95 microfilariae out of 100 passing through are killed 
by the spines, then nearly the entire volume must have been subjected 
to maceration. Wing beat frequencies range from approximately 150 to 
600 cycles per second in mosquitoes (Clements, 1963), and it is not 
unreasonable to suppose that the pumping mechanism oscillates similarly. 
Given a constant flow rate, damage rates would be a function of armature 
oscillation rat© and spine bed area. In the SEM photographs of Uchida 
(1979) » the bed is 25 um long by 30 pm wide and the shark-like teeth 
are in staggered rows spaced from 2-3 um apart. Thus, there is little 



117 

doubt that since nearly the entire volume ia processed as it is 
pimped by (since nearly all the microfilariae are damaged), the bed 
must close against the ventral pharyngeal valve completely. Given 
the small size of the bed, capable of macerating only about 750 pm , 
using the structures in Cx. pi plena pal lens as a model for the armature 
architecture and assuming that the flow rate through such a small 
orifice must be high, the oscillation rate of the valve imi3t also be 
very high. 

If the spine-to-spine distances mentioned above for Cx. pi pi ens 
pal lens are again used as a model, it is not inconceivable that the 
hemolysis of red cells is also due to the action of the armature, and, 
since no other sources of hemolysis could be found, it is tentatively 
concluded that this is indeed what is happening. Vacuolized chick 
red cells from Cx. nigripalpus were often seen in Giemsa stained thin 
3mears, and it is possible that this is a sign of injury and impending 
lysis. In the sugar-feeding mode experiments, hemolysis did not occur 
in groups where crop pH was near normal (Table II-Q), strongly suggesting 
that the 2 events are associated and the result of the same event. 

It should be mentioned that Uchida's (1979) photographs reveal, 
in addition to the dorsal pharyngeal valve, an array of spines and 
projections in the cibarium, and these could easily be involved in the 
wounding process. The results obtained with Aedes aeflypti here are in 
agreement with those of MoGreevy et al. , (1978), and Coluzzi and 
Trabucchi (1968), in that only rarely were microfilariae founded wounded 
in Ae. aegypti (or An. quadrimaculatus ) (Table 11-12), leading to the 
inevitable oonclusion the pharyngeal armature is essentially Inoperative 



118 

since these species do not possess a oibarial armature. Similarly, 
Coluzzi and Trabucchi (1968) found no damaged microfilariae in Ag . 
aegypti or An. quadrimacul atus . 

Conclusions 

The increase in the susceptibility of Cx. niffripalpus to 
infection by D. imiaitis produced by feeding the insects on glass bead- 
defibrinated infectious dog blood is evidence that supports the 
hypothesis, first advanced by Kartman (1953&) that hindrance of 
migration of microfilariae through the mosquito midgut to the target 
tissue plays a role in determining the characteristic vector-parasite 
relationship. However, this hindrance is not, as suggested by Kartman 
(1953a), associated with the loss of micro filarial vitality induced by 
exposure to salivary gland or midgut substances, but rather, as 
suggested by Nayar and Sauerman (l975)» manifests itself directly 
as the entrapment of microfilariae in an extremely rapidly clotting 
bloodmeal. Furthermore, this rapid clotting has been demonstrated, 
in this work, to be the result of the presence of a powerful coagulant, 
and it is not due to the absence of salivary gland anticoagulant, the 
presence of which is masked by the action of the coagulant. Therefore, 
the presence of the coagulant is a mechanism that contributes to the 
determination of the characteristic vector-parasite relationship. 

Micro filarial vitality loss is not related to hindrance of move- 
ment or to the presence of antiblastic factors, at least during the 
first few hours following feeding. Instead, it is the result of 
physical injury. The results of the present experiments are evidence 
in support of the hypothesis of Coluzzi and Trabucchi (1968), that the 



119 
wounds are the result of the action of the spiny dorsal pharyngeal 
valve ( bucco-pharyngeal armature), which must at times oscillate 
with high frequencies to produce the observed results. The previous 
work (Coluzzi and Trabucchi, 1968; Bryan et al. t 1974; McGreevy et al» . 
1978) has its basis in inferential comparative anatomy rather in 
additiomdeletion strategy, since in no instance, including the 
present one, has the armature been observed in action; the present 
work, however, seeks to augment the hypothesis through use of a 
strategy capitalizing on the differential feeding modes and meal 
processing sequences selected by a "switching mechanism" (Bay, 1954) » 
that probably evolved as a more efficient means of dealing with the 
vastly different chemical nature and function of the foods ingested; 
it was interesting to note, in this context, that saline enemas 
induced the secretion of a mucoid substance, possibly one of the 3 
secretions seen by Arnal (1950)t suggesting that secretion might be a 
function of pressure on the cells from the fluid. If this is true, 
then sugar meals would also produce the secretion, which would constitute 
a primitive and inefficient system. The evolution of the "switching 
mechanism" with detectors in the cibarium that would route a sugar 
meal to another organ, which could then release to the midgut small 
amounts intermit tan tly, the volume of which were too small to cause the 
necessary pressure, is clearly an advancement. Furthermore, if the 
"switching mechanism" did indeed "switch off" the oscillating armature 
as the results here suggest, this would constitute a deletion of the 
mechanism, albeit not of the structure, which in its open position, 
could operate as a sieving device. While these experiments cannot 
produce hard evidence such as direct observation of the traumatic event 
and therefore do not prove that the wounding is the work of the spiny 



120 

valve, the results are beat explained by that hypothesis and are 
supportive of the inferential conclusion. Additionally, it is also 
inferred that the associated hemolysis of erythrocytes and resultant 
oxyhemaglobin crystal formation, although not causal factors in either 
hindrance or trauma, are also the result of the action of the valve. 
The physical characteristics of the valve permit this, given the rapid 
oscillation speeds that mu3t be occurring. Benach and Crans (1975) 
allowed that hemolysis of frog erythrocytes might have been the work 
of the armature in Cx. territans . However, tests on salivary extracts 
during the course of these experiments indicated a possible "potentiator" 
role for thi3 secretion but the number of samples was too small to 
allow formation of definitive statements. Nevertheless, the "switching 
mechanism": dorsal pharyngeal valve combination is assigned the role of 
highly probable mechanism determining the characteristic vector- 
parasite relationship. 

When considered along with the unimpeded development of prelarvae 
in the target tissue of Cx. nigripalpus , these results constitute 
support for the hypothesis that the susceptibility level and host 
specificity are a function of the presence of a "gut barrier." In 
Cx . nigripalp us, this barrier has 2 components, entrapment and wounding, 
the mechanisms for which are coagulant-induced rapid clotting and 
"switch-mechanism"-induced wounding by the oscillations of the dorsal, 
pharyngeal valve. 

There is a confluence of the results with the general hypothesis 
established in insect-plant relationships, that host specificity is 
a function of the presence or absence of secondary host substances or 



121 

physical barriers that are capable of genetic manipulation (Frankel, 
1969); "this hypothesis hold3 that nutritional factors have only a 
small role in the actual determination of susceptibility level, a 
concept in part applied by others (Weathersby and McCall, 19685 
Weather3by et al» , 1971) to vector susceptibility problems in mosquito- 
malaria investigations, where, again, importance is assigned to "anti- 
blastic" (toxic) factors versus "atreptic" (nutritional) factors. The 
results reported here suggest that genetic manipulation of these 
mechanisms is possible, since they are variable entities. The actual 
events that generated genetically-selected differences in susceptibility 
patterns in Cule x pipiens hybrids infected with 3). immitis were not 
described by Kartman (1953&)» a ^d the transducer of the genetic message 
could bo valve function or coagulant presence, as both were variable 
entities. 



122 



Ae . aegypti Susceptibility Patterns— Background 

It is the purpose of this investigation to more completely 
characterize the responsible mechanism. Three sources of factors, the 
alimentary canal, Malpighian tubules, and hemolyrnch, are considered, 
although the evidence i3 suggestive of tubule involvement only. Within 
the tubules, the presence or absence of antiblaetic or atreptic sub- 
stances, the formation of membranous or pigmental capsules, and 
presence or absence of requisite physical conditions, are considered 
as possible sources of events, the result of which is arrest of 
development. The potential importance of knowledge of such a mechanism 
to the field of veterinary entomology and parasitology is obvious 
from the amount of work on-going in the field, and the similarities 
of this host-parasite system with those in which man is involved 
creates the potential for transference and interpretation of results 
within the public health purview, as pointed out by Kershaw, et al. t 
(1953); importantly the same phenomenon with Wuchereria and Brugia in 
the thoracic musculature of Aedes aegypti (MacDonald, 1962; Beckett 
and MacDonald, 1971; KacDonald and Ramachandran, 1965f) and in the fat 
body of Aedes ae gypti infected with Foleyella (Terwedow, 1973). It 
is also of importance to the emerging field of arthropod immunity. 



The Effects of Hindrance of Movement on Susceptibility of A&« Q8£TJ J}±L 
to D., immitis Infection 



The experiments by Kartman (I953 a ) with Ae. aegypti fed via 
membrane on anticoagulant-treated infectious dog blood produced a 
definite dislocation in the sequence of events characteristic of the 
"normal," unmanipulated vector-parasite relationship; the speed of 



123 



migration, as measured by the number of prelarvae in the tubules at 
24 after feeding, was distinctly greater than in controls fed directly 
on the dog. However, Kartman's (1953 a ) own results indicate that the 
event that actually determines the susceptibility level of a particular 
mosquito is manifested later, at approximately 72 , when shortening 
to the sausage stage should occur (Taylor, i960); this event, the 
arrest of development of prelarvae in the tubules, constitutes the 
major barrier encountered by D. immltis in this mosquito, and, that it 
is first demonstrable at 72 or greater after feeding does not prove 
that the actual determining event does not occur much earlier. Never- 
theless, Bince "host efficiency" is a function of arrested development 
in this mosquito (Kartman, 1953 a )f the problem remains that of deter- 
mining whether the dislocation induced by "deleting" microfilarial 
movement hindrance, through addition of anticoagulant, is associated 
with changes in susceptibility level, as measured by a change in the 
portion of arrested prelarvae; this was not addressed by Kartman 
(1953a), who discontinued his experiments after a 24" post-feed pre- 
larval count. It was the purpose of the present experiments to repeat 
the manipulation using addition of anticoagulant but to assay for 
developing larval forms at 120 after feeding, when more than enough 
time has elapsed for shortening to oocur. 



The portion of mosquitoes harboring developing forms was 
essentially unchanged by allowing them to feed on anitcoagul ant- 
treated or gla3s-bead defibrinated blood (Table 11-16); thus, increasing 
the rate of migration to the tubules ia not associated with an increase 
in group susceptibility to D. immitis . Circumvention of pre-raidgut 



124 

events by introduction of microfilariae via per ano (enema) or 
inoculation of Ae, aegypti freshly bloodfed on anticoagulant- 
treated chicken blood, likewise failed to alter the basic pattern, 
with arrest of development of prelarvae occurring in a majority of 
mosquitoes in the group. 

The Effect of Atreptic (Nutritional) Factors 

Although atreptic (nutritional) factors have not been generally 
considered to be determinants of susceptibility levels (Weathersby and 
McCall, 1968; Weathersby et al. , 1971), variation in the number of 
infective D. immitis larvae in groups of Ae. aegypti maintained on 
different carbohydrate sources was reported by otewart et al . , (1979)» 
suggesting that the absence of required factors might indeed influence 
the pattern of resistence. Preconditioning of groups of mosquitoes on 
a variety of nutrients, vitamins and minerals did not, however, result 
in consistent dislocations from the normal pattern (Table 11-16), and 
the same result was seen in groups of Ae. aegypti preconditioned with 
whole body extracts, prepared and administered by the same methods used 
by Weathersby e t al . , (1971)» or by hemocoelic infection with the extract, 
Although these experiments do not exhaustively treat the subject of 
absence of required factors, since the number of factors that could 
be "added" is far greater than those used here, these results, never- 
theless, do not support a contention that nutritional factors are 
involved in determination of susceptibility. 

The Effect of Antiblastic Factors 

The second major group of substances that could mediate the 
susceptibility level of Ae. aegypti to D. immitis , Uy inducing arrest 



125 



of development, falls into the category of "antiblastic" or toxic 
chemicals, produced by the host or host flora, inducing arrest by 
their "presence;" thus, the strategy employed to investigate their 
influence is one of "deletion." Since the melanization of prelarvae 
and even, rarely, sausages, has been reported by numerous investigators 
and was seen here (Figure II— 1 i), having been assigned a rather insig- 
nificant role by most, as characterized by Kartman (1953a), but with 
occasional studies indicating a more extensive role (Lindemann, 1977) i 
this phenomenon was investigated. The role of such substances as 
invertebrate defense mechanisms has been the subject of much investiga- 
tion (Poinar, 1973; Nappi, 1975). 

Dislocations from the baseline, untreated group pattern of 
susceptibility in Ae. ae/^ypti to D. immiti3 infection were not produced 
by either preconditioning with melanization inhibitors in the concentra- 
tions used here administered either per os in the maintenance 10/& 
sucrose solution or via thoracic injection (Table 11-16). Other 
experiments such as starvation and pre-blood feeding, designed to 
deplete antiblastic substances, were likewise unilluminating (Table 
11-16). While this evidence does not support the null hypothesis that 
melanization is responsible for arrest of development, the quantities 
used were based on levels found by others (Beresky and Hall, 1977) to 
be efficacious for inhibition of melanization in vivo , not on levels 
assayed and proven to work here. 

Interestingly, the inoculation of 24 old prelarvae from the 
tubules of Ae. aegypti into freshly bloodfed An. quadrimaculatus 
generated far more susceptibles than the baseline, indicating that 



126 




Figure 11-11. Melanization host response of Ae. aegypti to 
D. immitis in the malpighian tubule; in vitro ; x100. 



127 

not ail prelarvae have been arrested by this time, the infectious 
potential is less in prelarvae from +48 , which have much less 
motility at this time (Table 11-16). This is suggestive of a 
chronological factor, to be treated later. 

Interraittant feeding, initially used to reduce the microfilarial 
load on Ae . aeffypti fed directly on the infectious host, produced 
total dislocation of the vector-parasite relationship (Table II-16); 
the small control group size makes confirmation of this experimental 
approach necessary, and there are several possible explanations for the 
result. Although confirmed with several repetitions, a precise explana- 
tion must await further experiments; interference with hormone sensory 
systems is one possible explanation, since it should be emphasized 
that these mosquitoes were allowed to refeed continuously for JO min. 
for as long as the stated interval. Evidence to date does not support 
the hypothesis of hormonal involvement (Gwadz and Spielman, 1974)* 

Hemolymph Factors 

The hypothesis of Weatheroby (1965), based on his work with the 
Ae . aegypti ; Plasmodium g allinaceum vector-parasite relationship, that 
the antiblastic factors that determine immunity are systemic in character* 
distributed by the hemolymph, and are not confined to a particular 
tissue in the host, was tested for its applicability to the A®, aegypti ; 
D. immitis relationship by inoculating 20-30 D. immitis microfilariae 
into the hemocoel intrathoracically followed 120 later by total body 
dissection, after removel of the gut and tubules. If the arrest of 
development was the result of systemic antiblastic factors, exposure 
of microfilariae to the hemolymph could conceivably induce the phenomenon. 



128 



Unlike directly-fed baseline control group mosquitoes, no injected 
Ae. ae/crpti harbored microfilariae only (Table 11-17), which presents 
a pattern quite different than normal; in fact, a total of only 46 
microfilariae were found (8% of which were moribund), and they were 
always in association with prelarvao or presausages. The size of the 
presausage class is another distinct feature of the group and the 
greatest number of hemacyte-type host reactions (Figure II-i2)was in 
this group. Another provocative group is the sausages, which had the 
lowest percent of moribund individuals of the J> groups (4^o)» That 
there are so many mosquitoes harboring fully-formed larvae and in such 
numbers, does not support the position that factors which produce 
arrest of development are present in the hemolymph, although some 
growth in the absence of antagonists should not be unexpected in light 
of the results of Yoeli et al ., (1958), who found D. immitis sausages 
in waxmoths following infection of microfilariae. Microfilariae 
injected into Cx. nigripalpue fared less well than those in Ae. aegypti 
(Table 11-17), a result similar to that of Devaney and Howells (1979) 
working with mosquito cell lines. 

The Chronology of Arrest of Development 

Except for an ultras true tural study of Brugia pahangi prelarval 
development in Ae. aegypti thoracic musculature which demonstrated 
that abnormal development, perhaps the precursor of arrested develop- 
ment, occurs as soon as 12 after blood feeding (Lehane, 1978), the 
knowledge regarding the chronology of arrest has been general and does 
not specifically address the problem. Considering that arrest of 
development is the major determinant of the susceptibility level, it 



129 




Figure 11-12. Hemacyte host response of Ae. aegypti to 
D. immitis in the hemocoel, top center; thread-like 
structures are muscle fibers in the preparation; in vitro ; 
x100. 



130 

was considered necessary to investigate and characterize the com- 
ponent, if the mechanism were to be determined. 

Abnormal and dead prelarvae were found as early as 6 following 
ingestion (Table 11-18). By 8 , the portion of abnormal prelarvae 
had increased to 65% the substantial number of normal prelarvae at 
+24 (Table 11-18) complements the results of experiments involving 
injection of prelarvae (Table 11-16), although by +48 nearly 7<$ 
have been affected (Table 11-18). Thus, initiation of arrest could be 
taking place as early as 6 after bloodfeeding, but it nevertheless 
Is not simultaneous in action but spread out over a period of days 
(Table 11-18). 

Conclusions 

The phenomenon known as the arrest of development, of prelarvae 
of B. immitis in the tubules and Brugla sp. in the thoracic flight 
muscle, in Ae. a egypti , while being the major determinant of host 
efficiency (Karcman, 1953a), has not been closely studied or explained. 
It is known that undeveloped but living prelarvae of D. iramitis can 
exist for at least 2 weeks (Kartrnan, 1953a), and it has been suggested 
that arrest occurs because of failure of the prelarvae to attain, and 
retain, the quiescent, intracellular attitude requisite for sausage 
formation (Nayar and Sauerman, 1975 )» since the intracellular position 
is generally agreed upon to be the normal (non-pathological) location 
for prelarvae, within a short time of reaching the tubules (Taylor, 
I960; Weinstein, 1973), but more preoise descriptions of mechanisms to 
account for this were not presented. This was even the case in the 
one study which addressed "abnormal" development directly (Lehane, 1978), 



131 



explaining that abnormalities in cuticular formation in B. pahangi in 
A. aegypti occur as early as 12 after infection and are the first 
sign of refractoriness; the extensive cuticular reorganization, which 
must be completed if sausage formation and continued development are 
to be realized by the parasite, is somehow interfered with. Other 
hypotheses, although the biochemical of this phenomenon was not re- 
vealed, center around the phenomenon of membrane formation, since this 
is known to ocour in the D. immitis t Ae . aegypti relationship (Ashley, 
1972); and there is the possibility that lysosomal membrane formation 
i3 involved (Bradley and Nayar, 1980). However, the determination of 
whether the membrane is the product of the host or parasite can be an 
issue (Trager, 1974); and nothing has yet been published that indicates 
such membranes are not laid down on prelarvae already developing ab- 
normally. Likewise, the cuticular-abnormality problem (Lehane, 1976) 
fails to explain how prelarvae and normally developing later stages 
occur concurrently, in the same tubule and same insect. Little informa- 
tion regarding arrest has been gained thus far from in vitro cultiva- 
tion studies (Sawyer and Weinstein, 1963; Cupp, 1972) or other ultra- 
structural, studies on D. immitis (Kozek, 197 1). The evidence presented 
here supports the hypothesis that the events are determined in the 
tubules, not in the midgut or by hemolymph factors. There is the 
possibility of neurosecretory or other hormonal involvement, although 
there is evidence that hormones are not a factor (Gwadz and Spielman, 
1974), but the evidence in this instance is based on a rather small 

sample size and must await confirmation. Finally, the development of 
sausages in hemocoel throws into question the oft-asserted requirement 

for intracellular station for further development. 



132 



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133 



Table 1-2 Natural Potential Vectors of Canine Heartworm Disease 
in Vero Beach, Florida 



Species Collected and Species Found Positive 
for Dirofilaria imntitis Infection 











Species Captur 


3d/l>issected 


Positive 


% Positive 


Culex ni^ripalpus 


403 


2* 


0.5 


Cx. auinquefasciatus 


200 


: ! 


1.5 


Cx. melanoconion erraticus 


44 


o 





Cx. salinarius 


12 





G 


Cx. restuans 


2 








Aedes taeniorhynchus 


264 


8** 


3.0 


Ae. vexans 


27 
4 









Ae. aeffypti 





Ae. sollicitans 


2 








Anopheles auadrimaculatus 


11 








An. crucians 


3 








Psorophora columbiae 


50 








Coquillettidia perturbans 


:; 









* dead microfilariae found in bloodmeals from 1 specimen each of these 2 
species were presumed to be D. J nmitis based on length (average was 
315 u) and width (all were 6^8 ju . These cannot be designated "infected" 
mosquitoes, since microfilariae do not satisfy the criteria for that 
designation, since they have no potential for development unless they 
reach the Malpighian tubules. 

** one specimen from site 1 had 1 L3 in proboscis but was damaged in 
processing, preventing even presumptive identification. 



134 



Table 1-3 Natural Potential Vectors of Canine Heartworra Disease 

in Vero Beach, Florida 

Analysis of 16 Positive Female3 From 3 Species 



Species 



Mosquito Trap Trap Larval Larval Larval 
No. Site Type Location Type Number 



Culex quinquefasciatus 



Culex nigrlpalpus 



Aedes taeniorhynchus 



1 


l 


BT 


MP 


L] 




2 


2 


1 


BT 


MGlBM d 


f 

MF 


25 ( 


all dead) 


3 


? 


BT 


MT 


PL g 




5 


4 


1 


BT 


H:P e 


L3 




3 


] 


1 


BT 


H:P 


H 




2 


2 


3 


L? b 


MT 


L2 




2 


3 


2 


LT 


MG:BM 


MF 


10 ( 


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1 


1 


BO! 


H:P 


LJ 




2 


2 


1 


BT 


1 IT 


L1/L2 




2/2 


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1 


LT 


H:P 


13 




1 


4 


2 




MT/H;P 


L3 




1/3 


5 


1 


BT 


MT 


L2 




16 


6 


1 


LI- 


MI 


LI 




50 h 


7 


1 


LT 


!.? 


1 i 




8 1 


8 


1 


LT 


H:P 


L3 




1 


q 


1 


BT 


MT 


L2 




7 



"BT - bait trap; b LT - light trap; C MT - Malpighian tubules; TftJiBM - midguts 
bloodmeal; ^iP - head: proboscis; MF - Microfilariae (bloodmeal form; does 
not constitute infectious agents); g PL - prelarvae( identical to MF but con- 
stitutes infectious agents); with 15 melanized LI; with one melanized LI 



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BIOGRAPHICAL I 

Donald M. Sauerman, Jr., was born on June 29, 1940, in Elizabeth, 

New Jer3ey. After finishing primary school in Pelham Manor, New York, 
he attended secondary schools in Massachusetts, Ohio and Colorado, 
graduating cum laude from Fountain Valley School, Colorado Springs, 
Colorado. After attending the University of Connecticut, Storrs, Connec- 
ticut, from which he received his B.A. in 1964, he enrolled at Villanova 
University, Villanova, Pennsylvania, from which he received his M.S. in 
biology (1966)1 concurrently, he had become married to Rita Jeremics, 
formerly of Riga, Latvia. Following school, he obtained his first 
employment with the then Entomological Research Center, presently Florida 
Medical Entomology Laboratory, in Vero Eeach, Florida, where he is 
presently employed as &n entomologist. In 1974 and 1975 » he and his 
wife and two children, Marshall and Gretchen, moved to Gainesvillei Florida, 
the result of his having been awarded a year of leave with pay by the 
State of Florida, Health and Rehabilitative Services, to initiate the 
pursuit of his doctorate. He and his family are presently back in Vero 
Beach, where he is employed again by the Florida Medical Entomology 
Laboratory, while his wife is Indian River County Youth Guidance Volunteer 
Coordinator. 



1< ■: 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 




Jerry F. Butler, Chairman 

Professor of Entomology and Nematology 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 




Richard E. Bradley, Co-Chairman 
Professor of Parasitology 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 




Jai K. Nayar 
Medical Entomologist IV; 
Adjunct Associate Professor of 
Entomology and Nematology 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 



Donald W. Hall 

Professor of Entomology and Nematology 



I certify that I have read this study and that in my opinion 
it conforms to acceptable standards of scholarly presentation and 
is fully adequate, in scope and quality, as a dissertation for the 
degree of Doctor of Philosophy. 



Martin. D. Young /I (J 

Research Professor U 



This dissertation was submitted to the Graduate Faculty of the 
College of Agriculture and to the Graduate Council, and was accepted 
as partial fulfillment of the requirements for the degree of Doctor 
of Philosophy. 

December, 1980. 



Dean/jCollege of Ajjr'fcult 



Dean, Graduate School 



UNIVERSITY OF FLORIDA 



3 1262 08553 1993